Nicolai A. Kittan

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.

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.

Autoantibody-Mediated Bone Loss

In rheumatoid arthritis (RA), the presence of autoantibodies such as the rheumatoid factor and antibodies against citrullinated proteins is highly correlated with the severity of disease and bone loss. For many years, the involvement of autoantibodies in bone resorption has merely been attributed to enhanced tissue infiltration and the production of inflammatory cytokines that promote osteoclastogenesis. However, recent research provides evidence for a direct activation of osteoclasts and their precursors by autoantibodies, which is independent of inflammation. The depletion of B-cells with rituximab that substantially reduces autoantibody levels seems to be as effective as the well-established treatment with tumor necrosis factor-antagonists in RA patients that do not respond to methotrexate, highlighting the significance of autoantibodies for RA and bone loss.

STAT5-Induced Lunatic Fringe during Th2 Development Alters Delta-like 4–Mediated Th2 Cytokine Production in Respiratory Syncytial Virus–Exacerbated Airway Allergic Disease

Notch activation plays an important role in T cell development and mature T cell differentiation. In this study, we investigated the role of Notch activation in a mouse model of respiratory syncytial virus (RSV)–exacerbated allergic airway disease. During RSV exacerbation, in vivo neutralization of a specific Notch ligand, Delta-like ligand (Dll)-4, significantly decreased airway hyperreactivity, mucus production, and Th2 cytokines. Lunatic Fringe (Lfng), a glycosyltransferase that enhances Notch activation by Dll4, was increased during RSV exacerbation. Lfng loss of function in Th2-skewed cells inhibited Dll4-Notch activation and subsequent IL-4 production. Further knockdown of Lfng in T cells in CD4Cre+Lfngfl/fl mice showed reduced Th2 response and disease pathology during RSV exacerbation. Finally, we identified STAT5-binding cis-acting regulatory element activation as a critical driver of Lfng transcriptional activation. These data demonstrate that STAT5-dependent amplification of Notch-modifying Lfng augments Th2 response via Dll4 and is critical for amplifying viral exacerbation during allergic airway disease.

Inhibition of phosphodiesterase 4 (PDE4) reduces dermal fibrosis by interfering with the release of interleukin-6 from M2 macrophages

Objectives To investigate the disease-modifying effects of phosphodiesterase 4 (PDE4) inhibition in preclinical models of systemic sclerosis (SSc). Methods We studied the effects of PDE4 inhibition in a prevention and a treatment model of bleomycin-induced skin fibrosis, in the topoisomerase mouse model as well as in a model of sclerodermatous chronic graft-versus-host disease. To better understand the mode of action of PDE4 blockade in preclinical models of SSc, we investigated fibrosis-relevant mediators in fibroblasts and macrophages from healthy individuals and patients suffering from diffuse-cutaneous SSc on blockade of PDE4. Results Specific inhibition of PDE4 by rolipram and apremilast had potent antifibrotic effects in bleomycin-induced skin fibrosis models, in the topoisomerase I mouse model and in murine sclerodermatous chronic graft-versus-host disease. Fibroblasts were not the direct targets of the antifibrotic effects of PDE4 blockade. Reduced leucocyte infiltration in lesional skin on PDE4 blockade suggested an immune-mediated mechanism. Further analysis revealed that PDE4 inhibition decreased the differentiation of M2 macrophages and the release of several profibrotic cytokines, resulting in reduced fibroblast activation and collagen release. Within these profibrotic mediators, interleukin-6 appeared to play a central role. Conclusions PDE4 inhibition reduces inflammatory cell activity and the release of profibrotic cytokines from M2 macrophages, leading to decreased fibroblast activation and collagen release. Importantly, apremilast is already approved for the treatment of psoriasis and psoriatic arthritis. Therefore, PDE4 inhibitors might be further developed as potential antifibrotic therapies for patients with SSc. Our findings suggest that particularly patients with inflammation-driven fibrosis might benefit from PDE4 blockade.