To gain more insight into the mechanisms of particulate matter (PM)-induced adjuvant activity, we studied the kinetics of airway toxicity/inflammation and allergic sensitization to ovalbumin (OVA) in ...response to ultrafine carbon black particles (CBP). Mice were exposed intranasally to OVA alone or in combination with different concentrations of CBP. Airway toxicity and inflammation were assessed at days 4 and 8. Immune adjuvant effects were studied in the lung draining peribronchial lymph nodes (PBLN) at day 8. Antigen-specific IgE was measured at days 21 and 28, whereas allergic airway inflammation was studied after OVA challenges (day 28). Results show that a total dose of 200 μg CBP per mouse, but not 20 μg or 2 μg, induced immediate airway inflammation. This 200 μg CBP was the only dose that had immune adjuvant activity, by inducing enlargement of the PBLN and increasing OVA-specific production of Th2 cytokines (IL-4, IL-5, and IL-10). The immune adjuvant activity of 200 μg CBP dosing was further examined. Whereas increased OVA-specific IgE levels in serum on day 21 confirms systemic sensitization, this was further supported by allergic airway inflammation after challenges with OVA. Our data show a link between early airway toxicity and adjuvant effects of CBP. In addition, results indicate that local cytokine production early after exposure to CBP is predictive of allergic airway inflammation. In addition this model appears suitable for studying the role of airway toxicity, inflammation and other mechanisms of particle adjuvant activity, and predicting the adjuvant potential of different particles.
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 4
days 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-κB 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.
The thymotoxic organotin compounds di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) are known to induce apoptosis in vitro in rat thymocytes. They also affect macromolecular ...synthesis, inhibiting DNA synthesis and increasing RNA synthesis. Since these RNA molecules, likely to be involved in the initiation of the apoptotic process, have not been identified yet, the purpose of this research was to characterize by a cDNA macroarray the expression of genes involved in DBTC-induced apoptosis. We found that nur77 was rapidly transcripted in vitro following exposure of freshly isolated rat thymocytes to 3 μM DBTC. nur77 induction has also been observed in vivo after treatment of rats with apoptotic doses (60 mg/kg body wt) of DBTC. The products of nur77 are known to be involved in the apoptotic process, as nur77 is a transcription factor expressed in response to T-cell receptor-mediated apoptosis in immature T cells. Antisense oligonucleotide inhibition of nur77 expression prevented apoptosis induced by DBTC, supporting a role for nur77 in organotin-induced apoptotic cell death.
Abstract
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.
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.
The role of innate immune system in the onset of idiosyncratic drug‐induced liver injury is still matter of debate. Recent findings revealed how inflammation represents a key player in idiosyncratic drug‐induced liver injury. We evaluated the effects of trovafloxacin on the inflammation induced by tumor necrosis factor in mouse and found that trovafloxacin delays neutrophil and monocyte influx in mice livers. Considering the known neutrophil and monocyte anti‐inflammatory functions, the observed effect may hinder the regulation of inflammation contributing to the development of liver injury.
Immune-mediated drug hypersensitivity reactions (IDHR) may result from immuno-sensitization to a drug-induced neo-antigen. They rarely occur in patients and are usually not predicted preclinically ...using standard toxicity studies. To assess the potential of a drug to induce T-cell sensitization, trinitrophenyl (TNP)-Ficoll was used here as a bystander antigen in animal experiments. TNP-Ficoll will only elicit TNP-specific IgG antibodies in the presence of non-cognate T-cell help. Therefore, the presence of TNP-specific IgG antibodies after co-injection of drug and TNP-Ficoll was indicative of T-cell sensitization potential. This TNP-Ficoll-approach was used here to characterize T-cell help induced by oral exposure to diclofenac (DF) or carbamazepine (CMZ). DF or CMZ was administered orally to BALB/c mice and after 3 w, the mice were challenged in a hind paw with TNP-Ficoll and a dose of the drug that by itself does only elicit a sub-optimal popliteal lymph node assay (PLNA) response. T-cell-dependent responses were then evaluated in paw-draining popliteal lymph nodes (PLN). Also, shortly after oral exposure, mesenteric lymph nodes (MLN) were excised for evaluation of local responses. Both drugs were able to increase PLN cellularity and TNP-specific IgG
1
production after challenge. Both DF and CMZ stimulated CD4
+
and CD8
+
T-cells and caused shifts of the subsets toward an effector phenotype. DF, but not CMZ, appeared to stimulate interferon (IFN)-γ production. Remarkably, depletion of CD8
+
, but not CD4
+
, T-cells reduced TNP-specific IgG
1
production, and was more pronounced in CMZ- than in DF-exposed animals. Local responses in the MLN caused by DF or CMZ also showed shifts of CD4
+
and CD8
+
-cells toward a memory phenotype. Together, the data indicate that oral exposure to CMZ and DF differentially induced neo-antigen-specific T-cell reactions in the PLNA.
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.
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-formylindolo3,2-bcarbazole (FICZ), β-naphthoflavone (β-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, β-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, β-NF, and 6-MCDF on AhR activation in vitro. TCDD, but not FICZ, β-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, β-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, β-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, β-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.
Immune-mediated drug hypersensitivity reactions are important causes of black box warnings and drug withdrawals. Despite the high demand for preclinical screening tools, no validated in vitro or in ...vivo models are available. In the current study, we used a previously described oral administration model using trinitrophenyl-ovalbumin (TNP-OVA) as an antigen to report immuno-adjuvating effects of the analgesic drug acetaminophen (APAP) and its nonhepatotoxic regioisomer 3'-hydroxyacetanilide (AMAP), the antibiotic ofloxacin (OFLX), the antiepileptic drug carbamazepine (CMZ), and the antidiabetic drug metformin (MET). Furthermore, APAP and AMAP were tested in a popliteal lymph node assay (PLNA) combined with TNP-OVA as reporter antigen (RA). C3H/HeOuJ mice were dosed by oral gavage with diclofenac (DF), APAP, AMAP, OFLX, MET, or CMZ. On the first exposure day, the mice received an ip injection with TNP-OVA. Fifteen days later, they were ear challenged with TNP-OVA and delayed-type hypersensitivity (DTH) responses were assessed 24 h later. One week after challenge, the ear-draining lymph node was removed and TNP-specific antibody-secreting cells were determined. DF, APAP, CMZ, and OFLX showed a significant increase in DTH responses to ear injection with TNP-OVA, whereas AMAP and MET did not. C57BL/6 mice were slightly less responsive to APAP and DF after oral gavage, and importantly both AMAP and APAP were negative in the RA-PLNA. The present work shows that the oral exposure model using RA and the RA-PLNA may serve to screen the immune-adjuvant potential of new chemical entities during preclinical drug development.
We compared the effects of supplementing with vitamins A, C, and E, selenium, and zinc on a range of innate and specific T-helper 1 (Th1) and Th2-driven adaptive immune responses.
BALB/c mice were ...fed semi-purified AIN93 diets and randomly assigned to receive a diet supplemented with 120 mg/kg of vitamin A, 2500 mg/kg of vitamin C, 1000 mg/kg of vitamin E, 2 mg/kg of selenium, and 500 mg/kg of zinc (
n = 15/group). After 4 wk of supplementation, mice were sensitized by topical application of di-nitro-chlorobenzene (DNCB); 2 wk later mice were challenged; and 5 d later they were killed to assess the effect on a range of innate responses (phagocytic activity, oxidative burst and tumor necrosis factor-α), adaptive Th1-driven responses (delayed-type hypersensitivity, DNCB-specific immunoglobulin Ig G2a and IgG2b, and interferon-γ IFN-γ), and adaptive Th2-driven responses (DNCB-specific IgE and IgG1 and interleukin-4 IL-4).
Immune function was affected only in the vitamin A group. These mice gained less weight and were less capable of resolving the inflammatory response elicited during sensitization. The oxidative burst of blood cells was increased, but production of IFN-γ and IL-4 and the ratio of IFN-γ to IL-4 were markedly depressed. In concordance with the latter result, production of Th1-driven IgG2a antibodies was decreased, whereas Th2-driven isotypes were not affected (IgG1, IgE) and mucosal IgA was increased.
These findings confirmed that supplementary amounts of vitamin A above dietary requirements enhance inflammatory responses accompanied by decreased Th1 and increased mucosal responses. However, supplementation of these sufficiently fed, non-stressed, young adult mice with vitamins C and E, selenium, or zinc had no effect on immune function. We speculate that using this model in aged, physiologically, or nutritionally stressed mice may provide outcomes more similar to those in sensitive human populations. If so, this would improve the usefulness of the model to assess, characterize, and rank effects of foods or nutrients on a range of immune functions, including Th1/Th2 polarization.
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