Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis.

BACKGROUND: Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. METHODS: We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. RESULTS: Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. CONCLUSIONS: Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Cytokine and chemokine production by human pancreatic islets upon enterovirus infection.

Enteroviruses of the human enterovirus B species (HEV-Bs) (e.g., coxsackie B viruses [CVBs] and echoviruses) have been implicated as environmental factors that trigger/accelerate type 1 diabetes, but the underlying mechanism remains elusive. The aim of this study was to gain insight into the cytokines and chemokines that are produced by human pancreatic islets upon infection with CVBs. To this end, we studied the response of human islets of Langerhans upon mock or CVB3 infection. Using quantitative PCR, we showed that upon CVB3 infection, transcription of interferon (IFN), IFN-stimulated genes, and inflammatory genes was induced. Analysis of secreted cytokines and chemokines by Luminex technology confirmed production and secretion of proinflammatory cytokines (e.g., interleukin [IL]-6 and tumor necrosis factor-α) as well as various chemotactic proteins, such as IFN-γ-induced protein 10, macrophage inflammatory protein (MIP)-1α, MIP-1ß, and IL-8. Infection with other HEV-Bs induced similar responses, yet their extent depended on replication efficiency. Ultra violet-inactivated CVB3 did not induce any response, suggesting that virus replication is a prerequisite for antiviral responses. Our data represent the first comprehensive overview of inflammatory mediators that are secreted by human islets of Langerhans upon CVB infection and may shed light on the role of enteroviruses in type 1 diabetes pathogenesis.

TLR2 promotes Th2/Th17 responses via TLR4 and TLR7/8 by abrogating the type I IFN amplification loop.

TLR2 plays an important role in the removal of Gram-positive bacteria; contrastingly, it also appears to have important protective effects against unrestrained inflammation and subsequent organ injury during infection and autoimmunity. We hypothesized that TLR2 tunes the phenotype of dendritic cells (DCs) activated through other TLRs, thereby fulfilling a crucial role in the modulation of the immune response. TLR2 potently inhibited TLR4- and TLR7/8-induced cytokine production by human DCs. The inhibitory effect of TLR2 on the release of TNF-alpha but not of IL-12p70 was mediated by PI3K. TLR2 inhibits the production of IL-12p70 by dampening the type 1 IFN amplification loop. When DCs were triggered with the potent synergistic combination of LPS (TLR4) and R848 (TLR7/8) in conjunction with a TLR2 ligand, a clear shift to more Th2- and Th17-prone responses in the naive and memory T cell subpopulations was observed. This shift in T cell responses was inherent to the inability of TLR2-stimulated DCs to produce IL-12p70 and was dependent on the production of IL-1 and IL-6.

The inhibitory Fc gamma IIb receptor dampens TLR4-mediated immune responses and is selectively up-regulated on dendritic cells from rheumatoid arthritis patients with quiescent disease.

Rheumatoid arthritis (RA) is a common autoimmune disease leading to profound disability and premature death. Although a role for FcgammaRs and TLRs is accepted, their precise involvement remains to be elucidated. FcgammaRIIb is an inhibitory FcR important in the maintenance of tolerance. We hypothesized that the inhibitory FcgammaRIIb inhibits TLR responses on monocyte-derived dendritic cells (DC) and serves as a counterregulatory mechanism to dampen inflammation, and we surmised that this mechanism might be defective in RA. The expression of the inhibitory FcgammaRIIb was found to be significantly higher on DCs from RA patients having low RA disease activity in the absence of treatment with antirheumatic drugs. The expression of activating FcgammaRs was similarly distributed among all RA patients and healthy controls. Intriguingly, only DCs with a high expression of FcgammaRIIb were able to inhibit TLR4-mediated secretion of proinflammatory cytokines when stimulated with immune complexes. In addition, when these DCs were coincubated with the combination of a TLR4 agonist and immune complexes, a markedly inhibited T cell proliferation was apparent, regulatory T cell development was promoted, and T cells were primed to produce high levels of IL-13 compared with stimulation of the DCs with the TLR4 agonist alone. Blocking FcgammaRIIb with specific Abs fully abrogated these effects demonstrating the full dependence on the inhibitory FcgammaRIIb in the induction of these phenomena. This TLR4-FcgammaRIIb interaction was shown to dependent on the PI3K and Akt pathway.

Human mitochondrial complex I assembly is mediated by NDUFAF1.

Complex I (NADH:ubiquinone oxidoreductase) is the largest multiprotein enzyme of the oxidative phosphorylation system. Its assembly in human cells is poorly understood and no proteins assisting this process have yet been described. A good candidate is NDUFAF1, the human homologue of Neurospora crassa complex I chaperone CIA30. Here, we demonstrate that NDUFAF1 is a mitochondrial protein that is involved in the complex I assembly process. Modulating the intramitochondrial amount of NDUFAF1 by knocking down its expression using RNA interference leads to a reduced amount and activity of complex I. NDUFAF1 is associated to two complexes of 600 and 700 kDa in size of which the relative distribution is altered in two complex I deficient patients. Analysis of NDUFAF1 expression in a conditional complex I assembly system shows that the 700 kDa complex may represent a key step in the complex I assembly process. Based on these data, we propose that NDUFAF1 is an important protein for the assembly/stability of complex I.

Human mitochondrial complex I assembles through the combination of evolutionary conserved modules: a framework to interpret complex I deficiencies.

With 46 subunits, human mitochondrial complex I is the largest enzyme of the oxidative phosphorylation system. We have studied the assembly of complex I in cultured human cells. This will provide essential information about the nature of complex I deficiencies and will enhance our understanding of mitochondrial disease mechanisms. We have found that 143B206 rho zero cells, not containing mitochondrial DNA, are still able to form complex I subcomplexes. To further address the nature of these subcomplexes, we depleted 143B osteosarcoma cells of complex I by inhibiting mitochondrial protein translation with doxycycline. After removing this drug, complex I formation resumes and assembly intermediates were observed by two-dimensional blue native electrophoresis. Analysis of the observed subcomplexes indicates that assembly of human complex I is a semi-sequential process in which different preassembled subcomplexes are joined to form a fully assembled complex. The membrane part of the complex is formed in distinct steps. The B17 subunit is part of a subcomplex to which ND1, ND6 and PSST are subsequently added. This is bound to a hydrophilic subcomplex containing the 30 and 49 kDa subunits, to which a subcomplex including the 39 kDa subunit is incorporated, and later on the 18 and 24 kDa subunits. At a later stage more subunits, including the 15 kDa, are added and holo-complex I is formed. Our results suggest that human complex I assembly resembles that of Neurospora crassa, in which a membrane arm is formed and assembled to a preformed peripheral arm, and support ideas about modular evolution.

Pharmacokinetics and immunological aspects of a phase Ib study with intratumoral administration of recombinant human interleukin-12 in patients with head and neck squamous cell carcinoma: a decrease of T-bet in peripheral blood mononuclear cells.

The aim of this study was to evaluate the tolerability of intratumoral administered recombinant human interleukin-12 (rhIL-12) in patients with head and neck squamous cell carcinoma. Six patients were treated once a week at two dose levels of 100 or 300 ng/kg, respectively, up to 24 weeks. The primary end point was to assess the toxicity and safety of intratumoral injected rhIL-12 in head and neck squamous cell carcinoma patients; the pharmacokinetics and pharmacodynamics of rhIL-12 and any evidence of antitumor effect were also determined. Toxicity was mild, with prolonged grade 4 lymphopenia observed in only one patient. No dose-limiting toxicities occurred. In all six patients, the rhIL-12 was detectable in plasma within 30 min. Significant reductions in absolute number of peripheral blood lymphocytes and all lymphocyte subsets, especially cytotoxic T cells and natural killer cells, were observed that were maximal between 12 and 24 h. Maximal plasma concentrations of IFN-gamma and IL-10 were detected after 12 h. A real-time semiquantitative PCR analysis in peripheral blood mononuclear cells showed a mean increase of mRNA encoding IFN-gamma of 2.2 times relative to the pretreatment sample. An unexpected, significant decrease of 80% in T-bet mRNA, a T-helper 1 transcription factor, was detected after 12 h, with normalization after 48-72 h. No complete or partial responses were observed. In one patient, a 40% regression of a tumor lesion was noted. In conclusion, rhIL-12 at these dose levels and schedule was well tolerated and resulted in measurable immunological responses.