Dose-dependent inhibition of demyelination and microglia activation by IVIG.

OBJECTIVE: Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Clinical trials of IVIG for multiple sclerosis, using diverse dose regimens, yielded controversial results. The aim of this study is to dissect IVIG effector mechanisms on demyelination in an ex vivo model of the central nervous system (CNS)-immune interface. METHODS: Using organotypic cerebellar slice cultures (OSC) from transgenic mice expressing green fluorescent protein (GFP) in oligodendrocytes/myelin, we induced extensive immune-mediated demyelination and oligodendrocyte loss with an antibody specific for myelin oligodendrocyte glycoprotein (MOG) and complement. Protective IVIG effects were assessed by live imaging of GFP expression, confocal microscopy, immunohistochemistry, gene expression analysis and flow cytometry. RESULTS: IVIG protected OSC from demyelination in a dose-dependent manner, which was at least partly attributed to interference with complement-mediated oligodendroglia damage, while binding of the anti-MOG antibody was not prevented. Staining with anti-CD68 antibodies and flow cytometry confirmed that IVIG prevented microglia activation and oligodendrocyte death, respectively. Equimolar IVIG-derived Fab fragments or monoclonal IgG did not protect OSC, while Fc fragments derived from a polyclonal mixture of human IgG were at least as potent as intact IVIG. INTERPRETATION: Both intact IVIG and Fc fragments exert a dose-dependent protective effect on antibody-mediated CNS demyelination and microglia activation by interfering with the complement cascade and, presumably, interacting with local immune cells. Although this experimental model lacks blood-brain barrier and peripheral immune components, our findings warrant further studies on optimal dose finding and alternative modes of application to enhance local IVIG concentrations at the site of tissue damage.

Activation of the inflammasome by amorphous silica and TiO2 nanoparticles in murine dendritic cells.

Nanomaterials are increasingly used in various food applications. In particular, nanoparticulate amorphous SiO2 is already contained, e.g., in spices. Since intestinal dendritic cells (DC) could be critical targets for ingested particles, we compared the in vitro effects of amorphous silica nanoparticles with fine crystalline silica, and micron-sized with nano-sized TiO2 particles on DC. TiO2- and SiO2-nanoparticles, as well as crystalline silica led to an upregulation of MHC-II, CD80, and CD86 on DC. Furthermore, these particles activated the inflammasome, leading to significant IL-1ß-secretion in wild-type (WT) but not Caspase-1- or NLRP3-deficient mice. Silica nanoparticles and crystalline silica induced apoptosis, while TiO2 nanoparticles led to enhanced production of reactive oxygen species (ROS). Since amorphous silica and TiO2 nanoparticles had strong effects on the activation-status of DC, we suggest that nanoparticles, used as food additives, should be intensively studied in vitro and in vivo, to ensure their safety for the consumer.

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs stable establishment of oral tolerance in mice.

The toxic environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent immunomodulatory chemical. TCDD activates the aryl hydrocarbon receptor (AhR) and suppresses peripheral humoral and cellular adaptive immune responses. Though the major route of uptake is via food, little is known until now on the immunotoxic effects of TCDD on the gut-associated lymphoid tissue. We show here that AhR is strongly expressed along the small intestine, especially in intestinal epithelial cells (IEC). The AhR marker gene cyp1a1 is induced in IEC by oral TCDD exposure. We asked how TCDD affects oral tolerance, a unique function of mucosal immunity. C57BL/6 mice were injected with 10 µg/kg body weight TCDD and fed with ovalbumin (OVA) in a high-dose tolerization protocol. Mice were immunized and boosted with OVA on days 12, 23, and 55 after tolerization. Five of 14, 6 of 15, and 13 of 14 TCDD-treated mice generated OVA-specific immunoglobulin (Ig)G1 antibodies after the first, second, and third immunization with OVA, respectively. Only one mouse harbored anti-OVA IgG1 antibodies in the control group even after the third immunization with OVA. OVA-specific IgA in fecal samples of tolerized and TCDD-exposed mice could be detected at the levels of nontolerized mice, whereas completely absent in tolerant control mice. Correlated to this, we found in TCDD-treated mice an increase in interleukin-6 producing CD103+ dendritic cells (DC) present in the gut-draining mesenteric lymph nodes (MLN) and a small increase in the frequency of Th17 cells. Neither the frequencies nor the absolute numbers of immune cells in the lamina propria (LP) or in intraepithelial lymphocytes were changed by TCDD treatment. Our data not only have implications for food allergies in settings of environmental exposure but also raise concerns regarding the harmlessness of overdosing potential AhR agonist in food, which needs to be studied further.

Proinflammatory cytokines down-regulate intestinal selenoprotein P biosynthesis via NOS2 induction.

Selenoprotein P (SeP), serving as selenium transporter and extracellular antioxidant, is assumed to have a protective role in the gastrointestinal tract, which is particularly susceptible to oxidative damage. Decreased SeP mRNA levels have been found in colon cancer; however, information on the control of intestinal SeP biosynthesis is scarce. We analyzed SeP biosynthesis in human intestinal epithelial Caco-2 cells subject to differentiation from crypt- to villous-like enterocytes. In the course of Caco-2 cell differentiation, SeP mRNA expression and secretion increased concomitant with three regulators of SeP transcription: hepatocyte nuclear factor-4alpha, forkhead box class O1a, and peroxisomal proliferator-activated receptor-gamma coactivator 1alpha. Treatment of differentiated Caco-2 cells with the proinflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma caused a down-regulation of SeP biosynthesis, resulting from induction of nitric oxide synthase 2. These observations were corroborated by decreased SeP mRNA levels in the colon of dextran sodium sulfate-treated mice, an animal model of experimental colitis. We conclude that inflammation of the intestinal mucosa causes a decline in locally produced selenoprotein P in the colon that eventually may contribute to the emergence of inflammatory bowel disease-related colorectal cancer.

Suppression of cellular immunity by cord blood-derived unrestricted somatic stem cells is cytokine-dependent.

Unrestricted somatic stem cells (USSC) have the potential to differentiate into tissues derived from all three germinal layers and therefore hold promise for use in regenerative therapies. Furthermore, they have haematopoietic stromal activity, a characteristic that may be exploited to enhance haematopoietic engraftment. Both applications may require USSC to be used in an allogeneic, HLA-mismatched setting. We have therefore studied their in vitro interaction with cellular immunity. USSC showed no allostimulatory activity and caused only minimal inhibition of allogeneic T-cell responses. However, following pre-stimulation with IFNgamma and TNFalpha, they inhibited T-cell proliferation in an indoleamine 2, 3-dioxygenase-dependent manner and suppressed graft-versus-host type reactions. In addition, USSC inhibited DC maturation and function. This inhibition was overridden by stronger DC maturation signals provided by IL-1beta, IL-6, PGE(2) and TNFalpha compared to TNFalpha alone. Pre-stimulation of USSC with IFNgamma and TNFalpha had a similar effect: Inhibition of DC maturation was no longer observed. Thus, USSC are conditionally immunosuppressive, and IFNgamma and TNFalpha constitute a switch, which regulates their immunological properties. They either suppress T-cell responses in the presence of both cytokines or in their absence block DC differentiation and function. These activities may contribute to fine-tuning the immune system especially at sites of tissue damage in order to ensure appropriate differentiation of USSC and subsequent tissue repair. Therapeutically, they may help to protect USSC and possibly their progeny from immune rejection.

Monocyte derived dendritic cells generated by IFN-alpha acquire mature dendritic and natural killer cell properties as shown by gene expression analysis.

BACKGROUND: Dendritic cell (DC) vaccines can induce antitumor immune responses in patients with malignant diseases, while the most suitable DC culture conditions have not been established yet. In this study we compared monocyte derived human DC from conventional cultures containing GM-CSF and IL-4/TNF-alpha (IL-4/TNF-DC) with DC generated by the novel protocol using GM-CSF and IFN-alpha (IFN-DC). METHODS: To characterise the molecular differences of both DC preparations, gene expression profiling was performed using Affymetrix microarrays. The data were conformed on a protein level by immunophenotyping, and functional tests for T cell stimulation, migration and cytolytic activity were performed. RESULTS: Both methods resulted in CD11c+ CD86+ HLA-DR+ cells with a typical DC morphology that could efficiently stimulate T cells. But gene expression profiling revealed two distinct DC populations. Whereas IL-4/TNF-DC showed a higher expression of genes envolved in phagocytosis IFN-DC had higher RNA levels for markers of DC maturity and migration to the lymph nodes like DCLAMP, CCR7 and CD49d. This different orientation of both DC populations was confined by a 2.3 fold greater migration in transwell experiments (p = 0.01). Most interestingly, IFN-DC also showed higher RNA levels for markers of NK cells such as TRAIL, granzymes, KLRs and other NK cell receptors. On a protein level, intracytoplasmatic TRAIL and granzyme B were observed in 90% of IFN-DC. This translated into a cytolytic activity against K562 cells with a median specific lysis of 26% at high effector cell numbers as determined by propidium iodide uptake, whereas IL-4/TNF-DC did not induce any tumor cell lysis (p = 0.006). Thus, IFN-DC combined characteristics of mature DC and natural killer cells. CONCLUSION: Our results suggest that IFN-DC not only stimulate adaptive but also mediate innate antitumor immune responses. Therefore, IFN-DC should be evaluated in clinical vaccination trials. In particular, this could be relevant for patients with diseases responsive to a treatment with IFN-alpha such as Non-Hodgkin lymphoma or chronic myeloid leukemia.