ER stress abrogates the immunosuppressive effect of IL-10 on human macrophages through inhibition of STAT3 activation.

OBJECTIVE AND DESIGN: To determine whether ER stress affects the inhibitory pathways of the human immune system, particularly the immunosuppressive effect of IL-10 on macrophages. MATERIAL OR SUBJECTS: In vitro stimulation of human monocyte-derived macrophages. TREATMENT: Cells were stimulated with TLR ligands and IL-10, while ER stress was induced using thapsigargin or tunicamycin. METHODS: mRNA expression was determined using qPCR, while cytokine protein production was measured using ELISA. Protein expression of receptors and transcription factors was determined using flow cytometry. Student's t test was used for statistics. RESULTS: While under normal conditions IL-10 potently suppresses pro-inflammatory cytokine production by LPS-stimulated macrophages, we demonstrate that ER stress counteracts the immunosuppressive effects of IL-10, leading to increased pro-inflammatory cytokine production. We identified that ER stress directly interferes with IL-10R signaling by reducing STAT3 phosphorylation on Tyr705, which thereby inhibits the expression of SOCS3. Moreover, we show that ER stress also inhibits STAT3 activation induced by other receptors such as IL-6R. CONCLUSIONS: Combined, these data uncover a new general mechanism by which ER stress promotes inflammation. Considering its potential involvement in the pathogenesis of diseases such as Crohn's disease and spondyloarthritis, targeting of this mechanism may provide new opportunities to counteract inflammation.

Fc gamma receptor IIa suppresses type I and III interferon production by human myeloid immune cells.

Type I and type III interferons (IFNs) are fundamental for antiviral immunity, but prolonged expression is also detrimental to the host. Therefore, upon viral infection high levels of type I and III IFNs are followed by a strong and rapid decline. However, the mechanisms responsible for this suppression are still largely unknown. Here, we show that IgG opsonization of model viruses influenza and respiratory syncytial virus (RSV) strongly and selectively suppressed type I and III IFN production by various human antigen-presenting cells. This suppression was induced by selective inhibition of TLR, RIG-I-like receptor, and STING-dependent type I and III IFN gene transcription. Surprisingly, type I and III IFN suppression was mediated by Syk and PI3K independent inhibitory signaling via FcγRIIa, thereby identifying a novel non-canonical FcγRIIa pathway in myeloid cells. Together, these results indicate that IgG opsonization of viruses functions as a novel negative feedback mechanism in humans, which may play a role in the selective suppression of type I and III IFN responses during the late-phase of viral infections. In addition, activation of this pathway may be used as a tool to limit type I IFN-associated pathology.

FcαRI co-stimulation converts human intestinal CD103+ dendritic cells into pro-inflammatory cells through glycolytic reprogramming.

CD103+ dendritic cells (DC) are crucial for regulation of intestinal tolerance in humans. However, upon infection of the lamina propria this tolerogenic response is converted to an inflammatory response. Here we show that immunoglobulin A (IgA) immune complexes (IgA-IC), which are present after bacterial infection of the lamina propria, are important for the induction of inflammation by the human CD103+SIRPα+ DC subset. IgA-IC, by recognition through FcαRI, selectively amplify the production of proinflammatory cytokines TNF, IL-1ß and IL-23 by human CD103+ DCs. These cells then enhance inflammation by promoting Th17 responses and activating human intestinal innate lymphoid cells 3. Moreover, FcαRI-induced cytokine production is orchestrated via upregulation of cytokine translation and caspase-1 activation, which is dependent on glycolytic reprogramming mediated by kinases Syk, PI3K and TBK1-IKKε. Our data suggest that the formation of IgA-IC in the human intestine provides an environmental cue for the conversion of a tolerogenic to an inflammatory response.

New in vitro dermal absorption database and the prediction of dermal absorption under finite conditions for risk assessment purposes.

Most QSARs for dermal absorption predict the permeability coefficient, K(p), of a molecule, which is valid for infinite dose conditions. In practice, dermal exposure mostly occurs under finite dose conditions. Therefore, a simple model to predict finite dose dermal absorption from infinite dose data (K(p) and lag time) and the stratum corneum/water partition coefficient (K(SC,W)) was developed. To test the model, a series of in vitro dermal absorption experiments was performed under both infinite and finite dose conditions using acetic acid, benzoic acid, bis(2-ethylhexyl)phthalate, butoxyethanol, cortisone, decanol, diazinone, 2,4-dichlorophenol, ethacrynic acid, linolenic acid, octylparaben, oleic acid, propylparaben, salicylic acid and testosterone. For six substances, the predicted relative dermal absorption was not statistically different from the measured value. For all other substances, measured absorption was overpredicted by the model, but most of the overpredictions were still below the European default absorption value. In conclusion, our finite dose prediction model provides a useful and cost-effective estimate of dermal absorption, to be used in risk assessment for non-volatile substances dissolved in water at non-irritating concentrations.

Dermatokinetics of didecyldimethylammonium chloride and the influence of some commercial biocidal formulations on its dermal absorption in vitro.

The in vitro dermal absorption kinetics of didecyldimethylammonium chloride (DDAC) was studied after single and multiple exposure. In addition, the influence of biocidal formulations on the absorption of DDAC was investigated. Following dermal exposure to DDAC in aqueous solution, less than 0.5% of the applied dose reached the receptor fluid after 48h. The apparent permeability coefficient (K(p)) was 5+/-1cm/h x 10(-6) for concentrations <12.5mg/mL, and 12+/-3 cm/h x 10(-6) for concentrations >or=12.5 mg/mL, suggesting that DDAC decreases the skin barrier function. DDAC distributed readily into the stratum corneum, but the dermis appeared to be the main barrier for DDAC penetration. Multiple dosing of DDAC increased its flux across the skin, when applied in high concentrations (>11 mg/mL). However, the amount of DDAC reaching the receptor fluid remained low (<1% over a 48 h period). Selected biocidal formulations tended to reduce DDAC skin absorption. The degree of reduction appeared to be correlated to the amount of aldehydes present. Based on the comparison of the distribution of DDAC in full-thickness skin and epidermal membranes, we conclude that approximately one-third of the DDAC measured in the full-thickness membranes resides in the dermis. As a reasonable worst case assumption, this fraction could be considered systemically available when estimating the daily systemic body burden of DDAC.

Prediction of in vivo embryotoxic effect levels with a combination of in vitro studies and PBPK modelling.

The new EU legislations for chemicals (Registration, Evaluation and Authorization of Chemicals, REACH) and cosmetics (Seventh Amendment) stimulate the acceptance of in vitro and in silico approaches to test chemicals for their potential to cause reproductive effects. In the current study seven compounds with known in vivo developmental effects were tested in the embryonic stem cell test (EST). The EST correctly classified 5-fluorouracil, methotrexate, retinoic acid, 2-ethoxyacetic acid and 2-methoxyacetic acid for their in vivo embryotoxic potential. The toxicity of 2-methoxyethanol and 2-ethoxyethanol was underestimated due to a lack of metabolic capacity in the EST. This study further investigated the possibility to use in silico techniques to extrapolate in vitro effect concentrations determined in the EST to in vivo exposure levels. This approach was evaluated by comparing in silico predicted in vivo effect levels with effect levels measured in rodents. The in vivo effect levels of 2-methoxyethanol, 2-ethoxyethanol, methotrexate and retinoic acid were correctly predicted with in silico modelling. Contrary, in vivo embryotoxicity of 5-fluorouracil was overestimated following this approach. It is concluded that a combination of in vitro and in silico techniques appears to be a promising alternative test method for risk assessment of embryotoxic compounds.

Effects of single and repeated exposure to biocidal active substances on the barrier function of the skin in vitro.

The dermal route of exposure is important in worker exposure to biocidal products. Many biocidal active substances which are used on a daily basis may decrease the barrier function of the skin to a larger extent than current risk assessment practice addresses, due to possible skin effects of repeated exposure. The influence of repeated and single exposure to representative biocidal active substances on the skin barrier was investigated in vitro. The biocidal active substances selected were alkyldimethylbenzylammonium chloride (ADBAC), boric acid, deltamethrin, dimethyldidecylammonium chloride (DDAC), formaldehyde, permethrin, piperonyl butoxide, sodium bromide, and tebuconazole. Of these nine compounds, only the quaternary ammonium chlorides ADBAC and DDAC had a clear and consistent influence on skin permeability of the marker compounds tritiated water and [(14)C]propoxur. For these compounds, repeated exposure increased skin permeability more than single exposure. At high concentrations the difference between single and repeated exposure was quantitatively significant: repeated exposure to 300 mg/L ADBAC increased skin permeability two to threefold in comparison to single exposure. Therefore, single and repeated exposure to specific biocidal products may significantly increase skin permeability, especially when used undiluted.

Use of physicochemical calculation of pKa and CLogP to predict phospholipidosis-inducing potential: a case study with structurally related piperazines.

Several cationic amphiphilic compounds are known to induce phospholipidosis, a condition primarily characterized by excessive accumulation of phospholipids in different cell types, giving the affected cells a finely foamy appearance. Excessive storage of lamellar membranous intralysosomal inclusion bodies is the hallmark for phospholipidosis on the electron microscopic level. In case of alveolar phospholipidosis, foamy macrophages accumulate within the alveolar spaces of the lung. Based on such findings in a one-year toxicity study with gepirone in rats, we studied the molecular properties of this compound and compounds suspected of being phospholipidosis inducers by means of physicochemical calculations. Physicochemical molecular calculations of molecular weight, ClogP (partition coefficient octanol/water), logD at pH 7.4, and pKa were performed, for the cationic amphiphilic compounds chlorpromazine, amiodarone, imipramine, propranolol and fluoxetine, and for the structurally related compounds 1-phenylpiperazine (1-PHP), gepirone (and its major metabolites, 3-OH-gepirone and 1-pyrimidinylpiperazine [1-PP]), and buspirone. ClogP and calculated pKa cluster differently for the amphiphilic drugs compared to the chemical series of piperazines. In line with this analysis, lamellar inclusion bodies were found in an in vitro validation experiment in the human monoblastoid cell line U-937, incubated for 96 h at 10 microg/mL with cationic amphiphilic drugs (amiodarone, imipramine, or propranolol). No such lamellar inclusion bodies were seen for any of the compounds from the chemical series of piperazines including gepirone and its metabolites. The data presented support the use of simple physicochemical calculations of ClogP and pKa to discriminate rapidly between compounds suspected of being phospholipidosis inducers. Finally, the discriminative power of these physicochemical ClogP and pKa calculations to predict phospholipidosis-inducing potential was further validated by extension of the set of compounds.