Environmentally weathered polystyrene particles induce phenotypical and functional maturation of human monocyte-derived dendritic cells.

Micro- and nanoplastics (MNP) are ubiquitously present in the environment due to their high persistence and bioaccumulative properties. Humans get exposed to MNP via various routes and consequently, they will encounter dendritic cells (DC) which are antigen-presenting cells involved in regulating immune responses. The consequences of DC exposure to MNP are an important, yet understudied, cause of concern. Therefore, this study aimed to assess the uptake and effect of MNP in vitro by exposing human monocyte-derived dendritic cells (MoDC) to virgin and environmentally weathered polystyrene (PS) particles of different sizes (0.2, 1, and 10 µm), at different concentrations ranging from 1 to 100 µg/ml. The effects of these particles were examined by measuring co-stimulatory surface marker (i.e. CD83 and CD86) expression. In addition, T-cell proliferation was measured via a mixed-leukocyte reaction (MLR) assay. The results showed that MoDC were capable of absorbing PS particles, and this was facilitated by pre-incubation in heat-inactivated (HI) plasma. Furthermore, depending on their size, weathered PS particles in particular caused increased expression of CD83 and CD86 on MoDC. Lastly, weathered 0.2 µm PS particles were able to functionally activate MoDC, leading to an increase in T-cell activation. These in vitro data suggest that, depending on their size, weathered PS particles might act as an immunostimulating adjuvant, possibly leading to T-cell sensitization.

Enterocytes, fibroblasts and myeloid cells synergize in anti-bacterial and anti-viral pathways with IL22 as the central cytokine.

IL22 is an important cytokine involved in the intestinal defense mechanisms against microbiome. By using ileum-derived organoids, we show that the expression of anti-microbial peptides (AMPs) and anti-viral peptides (AVPs) can be induced by IL22. In addition, we identified a bacterial and a viral route, both leading to IL22 production by T cells, but via different pathways. Bacterial products, such as LPS, induce enterocyte-secreted SAA1, which triggers the secretion of IL6 in fibroblasts, and subsequently IL22 in T cells. This IL22 induction can then be enhanced by macrophage-derived TNFα in two ways: by enhancing the responsiveness of T cells to IL6 and by increasing the expression of IL6 by fibroblasts. Viral infections of intestinal cells induce IFNß1 and subsequently IL7. IFNß1 can induce the expression of IL6 in fibroblasts and the combined activity of IL6 and IL7 can then induce IL22 expression in T cells. We also show that IL22 reduces the expression of viral entry receptors (e.g. ACE2, TMPRSS2, DPP4, CD46 and TNFRSF14), increases the expression of anti-viral proteins (e.g. RSAD2, AOS, ISG20 and Mx1) and, consequently, reduces the viral infection of neighboring cells. Overall, our data indicates that IL22 contributes to the innate responses against both bacteria and viruses.

The hepatotoxic fluoroquinolone trovafloxacin disturbs TNF- and LPS-induced p65 nuclear translocation in vivo and in vitro.

Idiosyncratic drug-induced liver injury (IDILI) is a severe disease that cannot be detected during drug development. It has been shown that hepatotoxicity of some compounds associated with IDILI becomes apparent when these are combined in vivo and in vitro with LPS or TNF. Among these compounds trovafloxacin (TVX) induced apoptosis in the liver and increased pro-inflammatory cytokines in mice exposed to LPS/TNF. The hepatocyte survival and the cytokine release after TNF/LPS stimulation relies on a pulsatile activation of NF-κB. We set out to evaluate the dynamic activation of NF-κB in response to TVX + TNF or LPS models, both in mouse and human cells. Remarkably, TVX prolonged the first translocation of NF-κB induced by TNF both in vivo and in vitro. The prolonged p65 translocation caused by TVX was associated with an increased phosphorylation of IKK and MAPKs and accumulation of inhibitors of NF-κB such as IκBα and A20 in HepG2. Coherently, TVX suppressed further TNF-induced NF-κB translocations in HepG2 leading to decreased transcription of ICAM-1 and inhibitors of apoptosis. TVX prolonged LPS-induced NF-κB translocation in RAW264.7 macrophages increasing the secretion of TNF. In summary, this study presents new, relevant insights into the mechanism of TVX-induced liver injury underlining the resemblance between mouse and human models. In this study we convincingly show that regularly used toxicity models provide a coherent view of relevant pathways for IDILI. We propose that assessment of the kinetics of activation of NF-κB and MAPKs is an appropriate tool for the identification of hepatotoxic compounds during drug development.

Mouse strain differences in response to oral immunotherapy for peanut allergy.

BACKGROUND: Promising therapies for food allergy are emerging, mostly based on animal experimentation. However, different mouse strains are used, which may make it hard to compare experiments. The current study investigated whether the immunological differences between C3H/HeOuJ (C3H) and BALB/c mice lead to differences in efficacy of peanut-specific immunotherapy. METHODS: After sensitization using peanut extract (PE), C3H and BALB/c mice received oral immunotherapy (OIT) by intragastric dosing for three weeks. Hereafter, mice were exposed to PE via the intradermal, intragastric and intraperitoneal route, to determine allergic outcomes. Furthermore, PE-specific antibody and cytokine production were determined and the number of various immune cells at different time points during the study were measured. RESULTS: OIT protected C3H mice against anaphylaxis, whereas no anaphylaxis was seen in BALB/c mice. In contrast, OIT induced an increase in MMCP-1 levels in BALB/c mice but not in C3H mice. No effect of OIT on the acute allergic skin response was observed in either strain. Specific antibody responses showed similar patterns in both strains for IgA and IgG1. IgE levels were a tenfold higher in BALB/c mice and after the intragastric challenge (day 70) OIT-treated BALB/c mice showed induced IgE levels. Moreover, in C3H mice IgG2a levels were higher and increased in response to OIT and challenges. After the final challenge, but not at other timepoints MLN-derived lymphocytes from OIT-treated BALB/c mice produced less IL-13 and IL-5 compared to control-treated mice, whereas no differences were seen in case of C3H mice. CONCLUSIONS: Taken together, these results show that the C3H strain is more suitable to study clinical outcomes of OIT, whereas the BALB/c strain is more optimal to study T cell responses.

Trovafloxacin-Induced Liver Injury: Lack in Regulation of Inflammation by Inhibition of Nucleotide Release and Neutrophil Movement.

The fluoroquinolone trovafloxacin (TVX) is associated with a high risk of drug-induced liver injury (DILI). Although part of the liver damage by TVX+TNF relies on neutrophils, we have recently demonstrated that liver recruitment of monocytes and neutrophils is delayed by TVX. Here we show that the delayed leukocyte recruitment is caused by a combination of effects which are linked to the capacity of TVX to block the hemichannel pannexin 1. TVX inhibited find-me signal release in apoptotic HepG2 hepatocytes, decelerated freshly isolated human neutrophils toward IL-8 and f-MLF, and decreased the liver expression of ICAM-1. In blood of TVX+TNF-treated mice, we observed an accumulation of activated neutrophils despite an increased MIP-2 release by the liver. Depletion of monocytes and neutrophils caused increased serum concentrations of TNF, IL-6, and MIP-2 in TVX-treated mice as well as in mice treated with the fluoroquinolone levofloxacin, known to have a lower DILI-inducing profile. This supports the idea that early leukocyte recruitment regulates inflammation. In conclusion, disrupted regulation by leukocytes appears to constitute a fundamental step in the onset of TVX-induced liver injury, acting in concert with the capability of TVX to induce hepatocyte cell death. Interference of leukocyte-mediated regulation of inflammation represents a novel mechanism to explain the onset of DILI.

Dietary Supplementation with Nondigestible Oligosaccharides Reduces Allergic Symptoms and Supports Low Dose Oral Immunotherapy in a Peanut Allergy Mouse Model.

SCOPE: A major downside of oral immunotherapy (OIT) for food allergy is the risk of severe side effects. Non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS) reduce allergy development in murine models. Therefore, it is hypothesized that scFOS/lcFOS can also support the efficacy of OIT in a peanut allergy model. METHODS AND RESULTS: After sensitization to peanut extract (PE) using cholera toxin, C3H/HeOuJ mice are fed a 1% scFOS/lcFOS or control diet and receive OIT (1.5 or 15 mg PE). Hereafter, mice are exposed to PE via different routes to determine the safety and efficacy of treatment in clinical outcomes, PE-specific antibody production, and numbers of various immune cells. scFOS/lcFOS increases short-chain fatty acid levels in the caecum and reduce the acute allergic skin response and drop in body temperature after PE exposure. Interestingly, 15 mg and 1.5 mg OIT with scFOS/lcFOS induce protection against anaphylaxis, whereas 1.5 mg OIT alone does not. OIT, with or without scFOS/lcFOS, induces PE-specific immunoglobulin (Ig) IgG and IgA levels and increases CD103+ dendritic cells in the mesenteric lymph nodes. CONCLUSIONS: scFOS/lcFOS and scFOS/lcFOS combined with low dose OIT are able to protect against a peanut-allergic anaphylactic response.

Tissue influx of neutrophils and monocytes is delayed during development of trovafloxacin-induced tumor necrosis factor-dependent liver injury in mice.

Idiosyncratic drug-induced liver injury (iDILI) has a poorly understood pathogenesis. However, iDILI is often associated with inflammatory stress signals in human patients as well as animal models. Tumor necrosis factor (TNF) and neutrophils play a key role in onset of trovafloxacin (TVX)-induced iDILI, but the exact role of neutrophils and other leukocytes remains to be defined. We therefore set out to study the kinetics of immunological changes during the development of TVX-induced iDILI in the established murine model of acute liver injury induced by administration of TVX and TNF. Initially, TNF stimulated the appearance of leukocytes, in particular neutrophils, into the liver of TVX-treated mice, but even more so in control mice treated with the non-DILI inducing analogue levofloxacin (LVX) or saline as vehicle (Veh). This difference was apparent at 2 hours after TNF administration, but at 4 hours, the relative neutrophil amounts were reduced again in Veh- and LVX-treated mice whereas the amounts in TVX-treated mice remained at the same increased level as at 2 hours. The influx of monocytes/macrophages, which was unaffected in Veh- and LVX-treated mice was markedly reduced or even absent in TVX-treated mice. Unlike controls, mice receiving TVX + TNF display severe hepatotoxicity with clear pathology and apoptosis, coagulated hepatic vessels and increased alanine aminotransferase levels and interleukin 6/10 ratios. Findings indicate that TVX delays the acute influx of neutrophils and monocytes/macrophages. Considering their known anti-inflammatory functions, the disruption of influx of these innate immune cells may hamper the resolution of initial cytotoxic effects of TVX and thus contribute to liver injury development.

Indole-3-carbinol, a plant nutrient and AhR-Ligand precursor, supports oral tolerance against OVA and improves peanut allergy symptoms in mice.

In general, dietary antigens are tolerated by the gut associated immune system. Impairment of this so-called oral tolerance is a serious health risk. We have previously shown that activation of the ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) by the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects both oral tolerance and food allergy. In this study, we determine whether a common plant-derived, dietary AhR-ligand modulates oral tolerance as well. We therefore fed mice with indole-3-carbinole (I3C), an AhR ligand that is abundant in cruciferous plants. We show that several I3C metabolites were detectable in the serum after feeding, including the high-affinity ligand 3,3´-diindolylmethane (DIM). I3C feeding robustly induced the AhR-target gene CYP4501A1 in the intestine; I3C feeding also induced the aldh1 gene, whose product catalyzes the formation of retinoic acid (RA), an inducer of regulatory T cells. We then measured parameters indicating oral tolerance and severity of peanut-induced food allergy. In contrast to the tolerance-breaking effect of TCDD, feeding mice with chow containing 2 g/kg I3C lowered the serum anti-ovalbumin IgG1 response in an experimental oral tolerance protocol. Moreover, I3C feeding attenuated symptoms of peanut allergy. In conclusion, the dietary compound I3C can positively influence a vital immune function of the gut.

Oral exposure to immunostimulating drugs results in early changes in innate immune parameters in the spleen.

The development of immune-dependent drug hypersensitivity reactions (IDHR) is likely to involve activation of the innate immune system to stimulate neo-antigen specific T-cells. Previously it has been shown that, upon oral exposure to several drugs with immune-adjuvant capacity, mice developed T-cell-dependent responses to TNP-OVA. These results were indicative of the adjuvant potential of these drugs. The present study set out to evaluate the nature of this adjuvant potential by focusing on early immune changes in the spleen, by testing several drugs in the same experimental model. Mice were exposed to one or multiple oral doses of previously-tested drugs: the non-steroidal-anti-inflammatory drug (NSAID) diclofenac (DF), the analgesic acetaminophen (APAP), the anti-epileptic drug carbamazepine (CMZ) or the antibiotic ofloxacin (OFLX). Within 24 h after the final dosing, early innate and also adaptive immune parameters in the spleen were examined. In addition, liver tissue was also evaluated for damage. Exposure to APAP resulted in severe liver damage, increased levels of serum alanine aminotransferase (ALT) and local MIP-2 expression. DF exposure did not cause visible liver damage, but did increase liver weight. DF also elicited clear effects on splenic innate and adaptive immune cells, i.e. increased levels of NK cells and memory T-cells. Furthermore, an increase in plasma MIP-2 levels combined with an influx of neutrophils into the spleen was observed. OFLX and CMZ exposure resulted in increased liver weights, MIP-2 expression and up-regulation of co-stimulatory molecules on antigen-presenting cells (APC). The data suggested that multiple immune parameters were altered upon exposure to drugs known to elicit immunosensitization and that broad evaluation of immune changes in straightforward short-term animal models is needed to determine whether a drug may harbor the hazard to induce IDHR. The oral exposure approach as used here may be applied in the future as an immunotoxicological research tool in this type of evaluation.

DHA-rich tuna oil effectively suppresses allergic symptoms in mice allergic to whey or peanut.

BACKGROUND: Supplementation with long-chain n-3 polyunsaturated fatty acids (LCPUFAs) has been found to reduce the development of allergic disease. OBJECTIVE: The aim of this study was to compare the effectiveness of fish oil diets rich in eicosapentaenoic acid (20:5n-3; EPA) or docosahexaenoic acid (22:6n-3; DHA) in suppressing food allergic symptoms. METHODS: Mice were fed a control diet (10% soybean oil) or fish oil diet rich in EPA (4% soybean oil + 6% EPA oil containing 28.8% EPA and 13.7% DHA) or DHA (4% soybean oil + 6% DHA oil containing 7% EPA and 27.8% DHA), starting 14 d before and for 5 wk during oral sensitization with peanut extract (PE) or whey. Acute allergic skin responses, serum immunoglobulins (Igs), and mucosal mast cell protease-1 (mmcp-1) were assessed. Hyperimmune serum was transferred to naive recipient mice fed the different diets. RESULTS: The DHA diet effectively reduced the acute allergic skin response compared with the control or EPA diet in PE-allergic mice (control, 159 ± 15, or EPA, 129 ± 8, vs. DHA, 78 ± 7 µm; P < 0.0001 or P < 0.05, respectively). In contrast, both the DHA and EPA diets reduced the allergic skin response in whey allergic mice (control, 169 ± 9, vs. DHA, 91 ± 13, or EPA, 106 ± 14 µm; P < 0.001 or P < 0.01, respectively); however, only the DHA diet reduced mmcp-1 and whey-specific IgE and IgG1. The DHA and EPA diets also reduced the acute skin response in passively immunized mice. CONCLUSIONS: The DHA-rich fish oil diet reduced allergic sensitization to whey and allergic symptoms in both PE- and whey-allergic mice. These data suggest that DHA-rich fish oil is useful as an intervention to prevent or treat food allergy symptoms.

Non-dioxin-like AhR ligands in a mouse peanut allergy model.

Recently, we have shown that AhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses sensitization to peanut at least in part by inducing a functional shift toward CD4(+)CD25(+)Foxp3(+) T cells. Next to TCDD, numerous other AhR ligands have been described. In this study, we investigated the effect of three structurally different non-dioxin-like AhR ligands, e.g., 6-formylindolo[3,2-b]carbazole (FICZ), ß-naphthoflavone (ß-NF), and 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), on peanut sensitization. Female C57BL/6 mice were sensitized by administering peanut extract (PE) by gavage in the presence of cholera toxin. Before and during peanut sensitization, mice were treated with FICZ, ß-NF, or 6-MCDF. AhR gene transcription in duodenum and liver was investigated on day 5, even as the effect of these AhR ligands on CD4(+)CD25(+)Foxp3(+) T(reg) cells in spleen and mesenteric lymph nodes (MLNs). Mice treated with TCDD were included as a positive control. Furthermore, the murine reporter cell line H1G1.1c3 (CAFLUX) was used to investigate the possible role of metabolism of TCDD, FICZ, ß-NF, and 6-MCDF on AhR activation in vitro. TCDD, but not FICZ, ß-NF, and 6-MCDF, suppressed sensitization to peanut (measured by PE-specific IgE, IgG1, IgG2a and PE-induced interleukin (IL)-5, IL-10, IL-13, IL-17a, IL-22, and interferon-γ). In addition, FICZ, ß-NF, and 6-MCDF treatments less effectively induced AhR gene transcription (measured by gene expression of AhR, AhRR, CYP1A1, CYP1A2, CYP1B1) compared with TCDD-treated mice. Furthermore, FICZ, ß-NF and 6-MCDF did not increase the percentage of CD4(+)CD25(+)Foxp3(+) T(reg) cells in spleen and mesenteric lymph nodes compared with PE-sensitized mice, in contrast to TCDD. Inhibition of metabolism in vitro increased AhR activation. Together, these data shows that TCDD, but not FICZ, ß-NF, and 6-MCDF suppresses sensitization to peanut. Differences in metabolism, AhR binding and subsequent gene transcription might explain these findings and warrant further studies to investigate the role of the AhR in food allergic responses.

Intestinal alkaline phosphatase contributes to the reduction of severe intestinal epithelial damage.

Inflammatory bowel disease is characterized by chronic inflammation of the intestine and is accompanied by damage of the epithelial lining and by undesired immune responses towards enteric bacteria. It has been demonstrated that intestinal alkaline phosphatase (iAP) protects against the induction of inflammation, possibly due to dephosphorylation of lipopolysaccharide (LPS). The present study investigated the therapeutic potential of iAP in intestinal inflammation and epithelial damage. Intestinal epithelial damage was induced in C57BL/6 mice using detran sulfate sodium (DSS) and iAP was administered 4days after initial DSS exposure. Loss in body weight was significantly less in iAP-treated mice and accompanied with reduced colon damage (determined by combination of crypt loss, loss of goblet cells, oedema and infiltrations of neutrophils). Treatment with iAP was more effective in case of severe inflammation compared to situations of mild to moderate inflammation. Rectal administration of LPS into a moderate inflamed colon did not aggravate inflammation. Furthermore, soluble iAP did not lower LPS-induced nuclear factor-kappaB activation in epithelial cells in vitro but induction of cellular AP expression by butyrate resulted in decreased LPS response. In conclusion, the present study shows that oral iAP administration has beneficial effects in situations of severe intestinal epithelial damage, whereas in moderate inflammation endogenous iAP may be sufficient to counteract disease-aggravating effects of LPS. An approach including iAP treatment holds a therapeutic promise in case of severe inflammatory bowel disease.