Philipp Schuster

Impaired Plasmacytoid Dendritic Cell Innate Immune Responses in Patients with Herpes Virus-Associated Acute Retinal Necrosis

Plasmacytoid dendritic cells (PDC), the main producers of type I IFNs in the blood, are important for the recognition and control of viral and bacterial infections. Because several viruses induce IFN-α production, severe courses of herpes virus infections in nonimmunocompromised patients may be related to numerical or functional PDC deficits. To evaluate this hypothesis, PBMC and PDC were repeatedly isolated from nine patients with acute retinal necrosis (ARN), caused by herpes simplex or varicella zoster virus. The patients experienced meningitis/encephalitis and frequent infections in childhood (n = 2), recurrent herpes virus infections at unusual localizations (n = 2), ocular surgery (n = 1), infections (n = 4), and stress around ARN (n = 6). The median percentage of isolated PDC was significantly lower in patients compared with 18 age-matched healthy controls (p < 0.001), confirmed by FACS analysis using peripheral blood, and was extremely low during acute disease. PDC counts dropped in five controls suffering from respiratory infections or diarrhea. IFN-α production in PDC and PBMC exposed to different stimuli was significantly lower in patients than in controls (p < 0.05). Anergy to these stimuli was observed on four occasions, in particular during acute disease. PDC of patients showed up-regulated IFN regulatory factor-7 mRNA levels and evidence of in vivo activation (CD80) and maturation (CD83) (p < 0.05). CD8+ cell responses were significantly lower in patients vs controls (p = 0.04). These data support a risk factor model in which numerical and functional deficits in PDC-mediated innate immune responses contribute to an impaired control of latent herpes virus infections and subsequent development of ARN.

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.

CD4 Binding Affinity Determines Human Immunodeficiency Virus Type 1-Induced Alpha Interferon Production in Plasmacytoid Dendritic Cells

ABSTRACT Plasmacytoid dendritic cells (PDC) are major producers of type I interferons (IFN) in response to human immunodeficiency virus type 1 (HIV-1) infection. To better define the underlying mechanisms, we studied the magnitude of alpha IFN (IFN-α) induction by recombinant viruses containing changes in the Env protein that impair or disrupt CD4 binding or expressing primary env alleles with differential coreceptor tropism. We found that the CD4 binding affinity but not the viral coreceptor usage is critical for the attachment of autofluorescing HIV-1 to PDC and for subsequent IFN-α induction. Our results illustrate the importance of the gp120-CD4 interaction in determining HIV-1-induced immune stimulation via IFN-α production.

CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells

Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p<0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

Co-ordinated regulation of plasmacytoid dendritic cell surface receptors upon stimulation with herpes simplex virus type 1

Human plasmacytoid dendritic cells (PDC) are crucial for innate and adaptive immune responses against viral infections, mainly through production of type I interferons. Evidence is accumulating that PDC surface receptors play an important role in this process. To investigate the PDC phenotype in more detail, a chip‐based expression analysis of surface receptors was combined with respective flow cytometry data obtained from fresh PDC, PDC exposed to interleukin‐3 (IL‐3) and/or herpes simplex virus type 1 (HSV‐1). CD156b, CD229, CD305 and CD319 were newly identified on the surface of PDC, and CD180 was identified as a new intracellular antigen. After correction for multiple comparisons, a total of 33 receptors were found to be significantly regulated upon exposure to IL‐3, HSV‐1 or IL‐3 and HSV‐1. These were receptors involved in chemotaxis, antigen uptake, activation and maturation, migration, apoptosis, cytotoxicity and costimulation. Infectious and ultraviolet‐inactivated HSV‐1 did not differentially affect surface receptor regulation, consistent with the lack of productive virus infection in PDC, which was confirmed by HSV‐1 real‐time polymerase chain reaction and experiments involving autofluorescing HSV‐1 particles. Viral entry was mediated at least in part by endocytosis. Time–course experiments provided evidence of a co‐ordinated regulation of PDC surface markers, which play a specific role in different aspects of PDC function such as attraction to inflamed tissue, antigen recognition and subsequent migration to secondary lymphatic tissue. This knowledge can be used to investigate PDC surface receptor functions in interactions with other cells of the innate and adaptive immune system, particularly natural killer cells and cytotoxic T lymphocytes.

Chronic immune activation in HIV-1 infection contributes to reduced interferon alpha production via enhanced CD40:CD40 ligand interaction.

Although a signature of increased interferon (IFN-)alpha production is observed in HIV-1 infection, the response of circulating plasmacytoid dendritic cells (PDC) to Toll-like receptor ligand stimulation is substantially impaired. This functional PDC deficit, which we specifically observed in HIV-1 infected individuals with less than 500 CD4+ T cells/µl, is not well understood. We provide evidence that the peripheral IFN-alpha production in HIV-1 infection is actively suppressed by the enhanced interaction of CD40 ligand (CD40L), a member of the tumor necrosis factor family, and its receptor CD40, which are both upregulated upon immune activation. Plasma levels of soluble CD40L were significantly higher in untreated HIV-1 infected individuals (n = 52) than in subjects on long-term antiretroviral therapy (n = 62, p<0.03) and in uninfected control donors (n = 16, p<0.001). Concomitantly, cell-associated CD40L and the expression of the receptor CD40 on the PDC were significantly upregulated in HIV-1 infection (p<0.05). Soluble and cell-associated CD40L inhibited the PDC-derived IFN-alpha production by CpG oligodeoxynucleotides dose-dependently. This suppressive effect was observed at much lower, physiological CD40L concentrations in peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals compared to controls (p<0.05). The CpG-induced IFN-alpha production in PBMC of HIV-1 infected donors was directly correlated with PDC and CD4+ T cell counts, and inversely correlated with the viral loads (p<0.001). In HIV-1 infected donors with less than 500 CD4+ T cells/µl, the CpG-induced IFN-alpha production was significantly correlated with the percentage of CD40-expressing PDC and the level of CD40 expression on these cells (p<0.05), whereas CD40L plasma levels played a minor role. In addition, low-dose CD40L contributed to the enhanced production of interleukin 6 and 8 in PBMC of HIV-1 infected donors compared to controls. Our data support the conclusion that the chronic immune activation in HIV-1 infection impairs peripheral PDC innate immune responses at least in part via enhanced CD40:CD40L interactions.

Both plasmacytoid dendritic cells and monocytes stimulate natural killer cells early during human herpes simplex virus type 1 infections

Herpes simplex virus type 1 (HSV‐1), a member of the herpes virus family, is characterized by a short replication cycle, high cytopathogenicity and distinct neurotropism. Primary infection and reactivation may cause severe diseases in immunocompetent and immunosuppressed individuals. This study investigated the role of human plasmacytoid dendritic cells (pDC) in the activation of natural killer (NK) cells for the control of herpesviral infections. Within peripheral blood mononuclear cells, UV‐inactivated HSV‐1 and CpG‐A induced CD69 up‐regulation on NK cells, whereas infectious HSV‐1 was particularly active in inducing NK cell effector functions interferon‐γ (IFN‐γ) secretion and degranulation. The pDC‐derived IFN‐α significantly contributed to NK cell activation, as evident from neutralization and cell depletion experiments. In addition, monocyte‐derived tumour necrosis factor‐α (TNF‐α) induced after exposure to infectious HSV‐1 was found to stimulate IFN‐γ secretion. A minority of monocytes was shown to be non‐productively infected in experiments using fluorescently labelled viruses and quantitative PCR analyses. HSV‐1‐exposed monocytes up‐regulated classical HLA‐ABC and non‐classical HLA‐E molecules at the cell surface in an IFN‐α‐dependent manner, whereas stress molecules MICA/B were not induced. Notably, depletion of monocytes reduced NK cell effector functions induced by infectious HSV‐1 (P < 0·05). Altogether, our data suggest a model in which HSV‐1‐stimulated pDC and monocytes activate NK cells via secretion of IFN‐α and TNF‐α. In addition, infection of monocytes induces NK cell effector functions via TNF‐α‐dependent and TNF‐α‐independent mechanisms. Hence, pDC and monocytes, which are among the first cells infiltrating herpetic lesions, appear to have important bystander functions for NK cells to control these viral infections.

Differential Effects of P-Class Versus Other CpG Oligodeoxynucleotide Classes on the Impaired Innate Immunity of Plasmacytoid Dendritic Cells in HIV Type 1 Infection

Abstract Human plasmacytoid dendritic cells (PDC) are the major producers of type I interferons (IFN) after stimulation with CpG oligodeoxynucleotides (ODN). HIV-1-infected patients show a deficit in PDC numbers and function with progression of disease. CpG ODN appear to be attractive therapeutics to support the impaired innate immunity in HIV-1 infection. PDC counts, phenotype, and function were analyzed in 23 HIV-infected untreated individuals and 16 controls. Markers for migration (CCR7), activation (CD80), maturation (CD83), and endocytosis (BDCA2) were evaluated at baseline and 20 h after in vitro stimulation with class A, B, C, and P ODN. PDC counts and the expression of BDCA2 on these cells were significantly lower in HIV-1-infected subjects compared to controls (both p < 0.001). After stimulation with CpG ODN, CD80 and CD83 were upregulated to a similar extent in patients and controls, whereas CCR7 was upregulated more efficiently by CpG-P and CpG-C than CpG-A in HIV-1-infected individuals compared to controls. The IFN-alpha induction significantly differed for the CpG ODN classes (A > P > C > B) in patients and controls (p < 0.05). Functional PDC deficits in IFN-alpha and TNF-alpha induction were particularly evident in subjects with less than 500 CD4(+) cells/mul. CpG-P ODNs not only induced remarkable IFN-alpha production in patient PBMCs, but also significantly upregulated the antibacterial and antiviral CXC chemokine IP-10. In conclusion, PDC counts, phenotype, and function are significantly impaired in HIV-1-infected subjects. Optimized P-class ODN may be effective in reversing this innate immune defect, which should be further evaluated in vivo.

The Role of Plasmacytoid Dendritic Cells in Innate and Adaptive Immune Responses against Alpha Herpes Virus Infections

In 1999, two independent groups identified plasmacytoid dendritic cells (PDC) as major type I interferon- (IFN-) producing cells in the blood. Since then, evidence is accumulating that PDC are a multifunctional cell population effectively coordinating innate and adaptive immune responses. This paper focuses on the role of different immune cells and their interactions in the surveillance of alpha herpes virus infections, summarizes current knowledge on PDC surface receptors and their role in direct cell-cell contacts, and develops a risk factor model for the clinical implications of herpes simplex and varicella zoster virus reactivation. Data from studies involving knockout mice and cell-depletion experiments as well as human studies converge into a “spider web”, in which the direct and indirect crosstalk between many cell populations tightly controls acute, latent, and recurrent alpha herpes virus infections. Notably, cells involved in innate immune regulations appear to shape adaptive immune responses more extensively than previously thought.

A subset of human plasmacytoid dendritic cells expresses CD8α upon exposure to herpes simplex virus type 1

Classical and plasmacytoid dendritic cells (DC) play important roles in the defense against murine and human infections with herpes simplex virus (HSV). So far, CD8α expression has only been reported for murine DC. CD8α+ DC have prominent cross-presenting activities, which are enhanced by murine CD8α+ PDC. The human orthologue of murine CD8α+ DC, the CD141 (BDCA3)+ DC, mainly cross-present after TLR3 ligation. We report here the serendipitous finding that a subset of human PDC upregulates CD8α upon HSV-1 stimulation, as shown by gene array and flow cytometry analyses. CD8α, not CD8ß, was expressed upon exposure. Markers of activation, migration, and costimulation were upregulated on CD8α-expressing human PDC. In these cells, increased cytokine and chemokine levels were detected that enhance development and function of T, B, and NK cells, and recruit immature DC, monocytes, and Th1 cells, respectively. Altogether, human CD8α+ PDC exhibit a highly activated phenotype and appear to recruit other immune cells to the site of inflammation. Further studies will show whether CD8α-expressing PDC contribute to antigen cross-presentation, which may be important for immune defenses against HSV infections in vitro and in vivo.