No evidence of the role of early chemical exposure in the development of ß-cell autoimmunity.

Exposure to environmental chemicals can modulate the developing immune system, but its role in the pathogenesis of type 1 diabetes is largely unexplored. Our objective was to study the levels of circulating concentrations of environmental pollutants during the first years of life and their associations with the later risk of diabetes-predictive autoantibodies. From two birth-cohort studies including newborn infants with HLA-conferred susceptibility to type 1 diabetes (FINDIA and DIABIMMUNE), we identified case children with at least one biochemical diabetes-associated autoantibody (n = 30-40) and from one to four autoantibody-negative controls per each case child matched for age, gender, diabetes-related HLA-risk, delivery hospital, and, in FINDIA, also dietary intervention group. Plasma levels of 13 persistent organic pollutants and 14 per- and polyfluorinated substances were analyzed in cord blood and plasma samples taken at the age of 12 and 48 months. Both breastfeeding and the geographical living environment showed association with circulating concentrations of some of the chemicals. Breastfeeding-adjusted conditional logistic regression model showed association between decreased plasma HBC concentration at 12-month-old children and the appearance of diabetes-associated autoantibodies (HR, 0.989; 95% Cl, 0.978-1.000; P = 0.048). No association was found between the plasma chemical levels and the development of clinical type 1 diabetes. Our results do not support the view that exposure to the studied environmental chemicals during fetal life or early childhood is a significant risk factor for later development of ß-cell autoimmunity and type 1 diabetes.

Th1/Th17 plasticity is a marker of advanced ß cell autoimmunity and impaired glucose tolerance in humans.

Upregulation of IL-17 immunity and detrimental effects of IL-17 on human islets have been implicated in human type 1 diabetes. In animal models, the plasticity of Th1/Th17 cells contributes to the development of autoimmune diabetes. In this study, we demonstrate that the upregulation of the IL-17 pathway and Th1/Th17 plasticity in peripheral blood are markers of advanced ß cell autoimmunity and impaired ß cell function in human type 1 diabetes. Activated Th17 immunity was observed in the late stage of preclinical diabetes in children with ß cell autoimmunity and impaired glucose tolerance, but not in children with early ß cell autoimmunity. We found an increased ratio of IFN-γ/IL-17 expression in Th17 cells in children with advanced ß cell autoimmunity, which correlated with HbA1c and plasma glucose concentrations in an oral glucose tolerance test, and thus impaired ß cell function. Low expression of Helios was seen in Th17 cells, suggesting that Th1/Th17 cells are not converted thymus-derived regulatory T cells. Our results suggest that the development of Th1/Th17 plasticity may serve as a biomarker of disease progression from ß cell autoantibody positivity to type 1 diabetes. These data in human type 1 diabetes emphasize the role of Th1/Th17 plasticity as a potential contributor to tissue destruction in autoimmune conditions.

Interleukin-17 immunity in pediatric Crohn disease and ulcerative colitis.

OBJECTIVE: The present understanding of inflammatory bowel disease pathogenesis mainly relies on studies of adult patients. Therefore, we studied the balance between T-effector and regulatory cells in pediatric inflammatory bowel disease. METHODS: Quantitative polymerase chain reaction and immunohistochemistry served to quantify the expression of immunological markers in mucosal biopsies and flow cytometry analysis was used in peripheral blood mononuclear cells. RESULTS: Colonic interleukin (IL)-17+, IL-22, and IL-6 mRNA upregulation and increase in the number of colonic IL-17 cells were demonstrated in both Crohn disease (CD) and ulcerative colitis (UC). Likewise, colonic forkhead box P3 (FOXP3+) mRNA expression and the number of colonic FOXP3 cells were increased both in CD and in UC and were accompanied in CD also with increased numbers of FOXP3+CD25 High CD4 cells in peripheral blood. Ileal relation of IL-17/CD4 cells was increased only in CD. CONCLUSIONS: We showed activation of colonic IL-17/IL-22 axis and upregulation of FOXP3 to occur both in pediatric CD and in UC, indicating shared immunological characteristics. Upregulation of IL-17 was restricted to colon in UC, but existed in the ileum and in the colon in active CD.

Dendritic cells from Crohn's disease patients show aberrant STAT1 and STAT3 signaling.

Abnormalities of dendritic cells (DCs) and STAT proteins have been reported in Crohn's disease (CD). Studies on JAK/STAT signaling in DCs are, however, lacking in CD. We applied a flowcytometric single-cell-based phosphoepitope assay to evaluate STAT1 and STAT3 pathways in DC subsets from CD patients. In addition, circulating DC counts were determined, together with the activation-related immunophenotype. We found that IL-6- and IFN-α-induced STAT3 phosphorylation and IFN-α-induced STAT1 phosphorylation were impaired in plasmacytoid DCs (pDCs) from CD patients (P = 0.005, P = 0.013, and P = 0.006, respectively). In myeloid DCs (mDCs), IFN-α-induced STAT1 and STAT3 phosphorylation were attenuated (P<0.001 and P = 0.048, respectively), but IL-10-induced STAT3 phosphorylation was enhanced (P = 0.026). IFN-γ-induced STAT1 signaling was intact in both DC subtypes. Elevated plasma IL-6 levels were detected in CD (P = 0.004), which strongly correlated with disease activity (ρ = 0.690, P<0.001) but not with IL-6-induced STAT3 phosphorylation. The numbers of pDCs and BDCA3+ mDCs were decreased, and CD40 expression on CD1c+ mDCs was increased in CD. When elucidating the effect of IL-6 signaling on pDC function, we observed that IL-6 treatment of healthy donor pDCs affected the maturation of and modified the T-cell priming by pDCs, favoring Th2 over Th1 type of response and the expression of IL-10 in T cells. Our results implicate DC signaling in human CD. Reduced IL-6 responsiveness in pDCs, together with the attenuated IFN-α-induced signaling in both DC subtypes, may contribute to the immunological dysregulation in CD patients.

In Crohn's Disease, Anti-TNF-α Treatment Changes the Balance between Mucosal IL-17, FOXP3, and CD4 Cells.

Aim. In Crohn's disease (CD), anti-TNF-α treatment is a potent medication. We aimed to characterize the effect of anti-TNF-α treatment on T effector and regulatory cells. Material and Methods. We studied T-effector and regulatory cells on cellular and mRNA levels in intestinal biopsy samples from 13 Crohn's disease patient. Biopsies were obtained at baseline and 3 months after anti-TNF-α treatment, and from 14 inflammation-free control subjects. Results. Patients had higher numbers of ileal IL-17(+) and forkhead box P3 (FOXP3)(+) cells than did control subjects, both before ( P ≤ 0.001 and P ≤ 0.05, resp.) and after the anti-TNF-α treatment (P ≤ 0.01, P ≤ 0.01). Intestinal interferon-γ and IL-17 mRNA expression was higher in Crohn's disease and remained elevated after anti-TNF-α treatment. The ratio of IL-17(+) cells to CD4(+) cells decreased (P ≤ 0.05) and compared to baseline the ratio of IL-17(+) cells to FOXP3(+) was lower after treatment (P ≤ 0.05). Conclusions. TNF-α-blocking agents improved intestinal balance between IL-17(+) T-effector and regulatory T cells, although intestinal IL-17 upregulation remained elevated.

Altered phenotype of peripheral blood dendritic cells in pediatric type 1 diabetes.

OBJECTIVE: Dendritic cells (DCs) are largely responsible for the activation and fine-tuning of T-cell responses. Altered numbers of blood DCs have been reported in type 1 diabetes (T1D). We aimed at characterizing the less well-known phenotypic properties of DCs in T1D. RESEARCH DESIGN AND METHODS: In a case-control setting, samples from a total of 90 children were studied by flow cytometry or by quantitative real-time PCR (qPCR). RESULTS: We found decreased numbers of myeloid DCs (mDCs) (8.97 vs. 13.4 cells/µL, P = 0.009, n = 31) and plasmacytoid DCs (pDCs) (9.47 vs. 14.6 cells/µL, P = 0.018, n = 30) in recent-onset T1D. Using a panel of antibodies against functionally important DC markers, we detected a decreased expression of CC chemokine receptor 2 (CCR2) on mDCs (percentage above negative control, P = 0.002, n = 29) and pDCs (median intensity, P = 0.003, n = 30) from T1D patients. In an independent series of children, the reduced expression of CCR2 was confirmed by qPCR in isolated mDCs (P = 0.043, n = 20). Serum concentrations of CCR2 ligands monocyte chemotactic protein-1 and -3 did not differ between the groups. A trend for an enhanced responsiveness of the nuclear factor-κB pathway (P = 0.063, n = 39) was seen in mDCs from children with ß-cell autoantibodies, which is possibly related to the reduced CCR2 expression, since CCR2 on mDCs was downregulated by nuclear factor-κB-activating agents. CONCLUSIONS: Given the role of CCR2 in DC chemotaxis and in DC-elicited Th1 differentiation, our results may indicate a functionally important DC abnormality in T1D affecting the initiation and quality of immune responses.

Additive effects of enhanced ambient ultraviolet B radiation and increased temperature on immune function, growth and physiological condition of juvenile (parr) Atlantic Salmon, Salmo salar.

Climate change models predict increased ultraviolet B (UVB) radiation levels due to stratospheric ozone depletion and global warming. In order to study the impact of these two environmental stressors acting simultaneously on the physiology of fish, Atlantic salmon parr were exposed, for 8 weeks in outdoor tanks, to different combinations of UVB radiation (depleted and enhanced) and temperature (standard rearing temperature of 14 °C or 19 °C). The immune function (plasma IgM, lysozyme activity and complement bacteriolytic activity), growth (body weight) and physiological condition (haematocrit and plasma protein concentration) of the fish were determined. Increased UVB level, regardless of water temperature, had a negative effect on immune function parameters, growth and physiological condition. Higher temperature increased plasma IgM concentration but had a negative effect on complement bacteriolytic activity under both spectral treatments. Increased temperature, irrespective of UVB level, increased fish growth but negatively affected haematocrit and plasma protein. Exposing the fish to enhanced UVB at elevated temperature increased plasma IgM concentration and slightly improved growth. However, complement activity and physiological condition parameters decreased more than when the fish were exposed to each stressor separately. The changes were mainly additive; no interactive or synergistic effects were observed. The negative impact of multiple stressors on immune function, together with predicted increases in pathogen load in warmer waters resulting from global climate change, suggest an increased risk to diseases in fishes.

Serum immune-activation potency and response to anti-TNF-α therapy in Crohn's disease.

AIM: To study whether immune-activation stage in serum of adult Crohn's disease (CD) patients correlates with disease activity and with treatment response to anti-tumor necrosis factor-α (TNF-α) therapy. METHODS: Serum samples were obtained from 15 adult CD patients introduced to anti-TNF-α therapy. The individual stage of immune activation was studied applying our new in vitro assay, in which target cells (donor derived peripheral blood mononuclear cells) were cultured with patient serum and the T-cell activation induced by the patient serum was studied using a panel of markers for effector [interferon γ (IFNγ), interleukin (IL)-5] and regulatory T-cells [forkhead transcription factor 3 (FOXP3) and glucocorticoid-induced tumour necrosis factor receptor (GITR)]. The endoscopic disease activity was assessed with the Crohn's disease endoscopic index of severity (CDEIS) before and 3 mo after therapy with an anti-TNF-α agent. RESULTS: Low induction of FOXP3 and GITR in target cells cultured in the presence of patient serum was associated with high disease activity i.e. CDEIS assessed before therapy (r = -0.621, P = 0.013 and r = -0.625, P = 0.013, respectively). FOXP3 expression correlated inversely with pre-treatment erythrocyte sedimentation rate (r = -0.548, P = 0.034). Low serum induced FOXP3 (r = -0.600, P = 0.018) and GITR (r = -0.589, P = 0.021) expression and low IFNγ secretion from target cells (r = -0.538, P = 0.039) associated with treatment response detected as a decrease in CDEIS. CONCLUSION: The immune-activation potency in the patient serum prior to anti-TNF-α therapy reflected intestinal inflammation and the therapeutic response.

IL-17 immunity in human type 1 diabetes.

Th17 immunity has been shown to regulate autoimmune diabetes in mice. IL-17 neutralization prevented development of diabetes when given postinitiation of insulitis but not earlier, suggesting interference with the effector phase of the disease. Islet-cell Ag-specific Th17 cells converted into IFN-gamma-secreting Th1-like cells and caused diabetes in mice recipients. The role of IL-17 in human type 1 diabetes (T1D) is, however, not established. In this study, we show upregulation of Th17 immunity in peripheral blood T cells from children with T1D. This was characterized by increased IL-17 secretion and expression of IL-17, IL-22, and retinoic acid-related orphan receptor C isoform 2, but also FOXP3 transcripts upon T cell activation in vitro. Also, circulating memory CD4 cells from children with T1D showed the same pattern of IL-17, IL-22 and FOXP3 mRNA upregulation, indicating IL-17 pathway activation in vivo. IL-17-positive T cells appeared to be CD4(+) cells expressing TCR-alphabeta and CCR6, and a subpopulation showed coproduction of IFN-gamma. Given the Th17 immunity in T1D, we demonstrated that IL-17 had detrimental effects on human islet cells in vitro; it potentiated both inflammatory and proapoptotic responses. Our findings highlight the role of IL-17 immunity in the pathogenesis of human T1D and point to a potential therapeutic strategy.

Combined T regulatory cell and Th2 expression profile identifies children with cow's milk allergy.

The role of T regulatory cells in spontaneous recovery from cow's milk allergy (CMA) is unclear. We investigated the mRNA expression of 12 T-cell markers and the protein expression of CD4, CD25, CD127, FoxP3 after in vitro beta-lactoglobulin stimulation of peripheral blood mononuclear cells from children with persisting CMA (n=16), early recovery (n=20) or no atopy (n=21). Artificial neural networks with exhaustive search for all marker combinations revealed that markers FoxP3, Nfat-C2, IL-16 and GATA-3 distinguished patients with persisting CMA most accurately from other study groups. FoxP3 mRNA expression following beta-lactoglobulin stimulation was highest in children with persisting CMA. Also the FoxP3 intensity in CD4(+) CD25(high)CD127(low) cells was higher in children with CMA compared with non-atopic children. The expression profile of both Th2- and T regulatory cell-related genes thus reflects the clinical activity of CMA. Tolerance, in contrast, is not characterized by activation of circulating T regulatory cells.

New means to monitor the effect of glucocorticoid therapy in children.

AIM: To study the individual effects of glucocorticoid (GC) therapy on the state of immune activation in patient serum. METHODS: We developed a novel assay in which the effect of corticosteroid-treated patient serum on healthy donor peripheral blood mononuclear cells (target cells) was studied, with a panel of markers for effector [interferon (IFN)gamma and interleukin (IL)-5] and regulatory T cells (FOXP3 and glucocorticoid-induced tumor necrosis factor receptor, GITR). The study group comprised 19 children with inflammatory bowel disease. The individual effect of patient serum on target cells was analyzed prior to GC therapy and 2 wk later. RESULTS: The effect of GC therapy mediated by patient serum was seen as a decrease in the target cells expression of regulatory T-cell-related markers GITR (median suppression 24%, range of suppression 1%-63%, in 2 cases increase of 6% and 77%, P < 0.01 for mitogen-activated target cells) and FOXP3 (median suppression 33%, range of suppression 0%-79%, in one case an increase of 173%, P < 0.05 for resting cells), and secretion of IFNgamma [from a mean of 87 700 pg/mL (SD 33 900 pg/mL) to 60 900 pg/mL (SD 44 200 pg/mL) in mitogen-activated target cells, 13 of the cases showed a decrease, P < 0.01]. The total or weight-related prednisolone dose did not correlate with the patient-serum-induced changes in the target cell markers. CONCLUSION: GC response could be monitored at an individual level by studying the effect of patient serum on signaling pathways of target immune cells.

Long-term UVB irradiation affects the immune functions of carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss).

The effects of long-term, low-dose ultraviolet B (UVB) radiation on immune functions of two fish species representing different taxonomic groups, carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss), were assessed in this study. The fish were exposed to 7, 20 or 60 mJ cm(-2) UVB three times per week, for 6 weeks. In carp, UVB exposure affected the respiratory burst activity of blood and head kidney phagocytes, differential blood leukocyte counts and blood chemistry. Phytohemagglutinin (PHA)-stimulated in vitro proliferation responses of blood and head kidney lymphocytes, however, remained unchanged. Rainbow trout tolerated the irradiations with fewer alterations, but significant changes were detected in blood chemistry and hematocrits of the irradiated fish. These results indicate that long-term exposure to low doses of UVB induces immunomodulation in fish, and that there are species-specific differences in sensitivity to irradiation.

Combined effects of agricultural activity and parasites on biomarkers in the bullfrog, Rana catasbeiana.

Agricultural contaminants can have devastating impacts on amphibian survival and development, particularly considering their sensitivity to environmental perturbation. However, it is commonly overlooked that amphibians are infected with various parasites that can influence the overall health of the animal when exposed to a stressful environment. We investigated the interaction of agriculture and parasitism on the health of bullfrogs (Rana catesbeiana) in the field. Nine physiological and immunological biomarkers were related to naturally acquired parasite infections, along a gradient of agricultural activity. Most health biomarkers were affected by agriculture, parasitism, or both. Although bullfrogs residing in agricultural areas were infected with fewer parasite species, reflecting environmentally compromised ecosystems, certain persistent parasites interacted with agricultural disturbance to alter the physiology and immune competence of bullfrogs. The consequences of the combination for animal health highlight the importance of parasitism in ecotoxicological studies. Consideration of parasitism is warranted when evaluating the influence of anthropogenic disturbance on amphibian declines and environmental health.

Exposure to increased ambient ultraviolet B radiation has negative effects on growth, condition and immune function of juvenile Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) parr were exposed in two outdoor experiments, ranging in duration from 52 to 137 days, to spectral treatments: (1) natural sunlight (=present ambient UVB level), (2) solar radiation supplemented with enhanced UVB radiation from lamps simulating 20% or 8% stratospheric ozone loss or (3) UVB-depleted sunlight achieved by screening with Mylar-D film. The growth, condition and immune function of the salmon were quantified after treatments. Exposure to enhanced UVB radiation retarded growth, and decreased hematocrit value and plasma protein concentration. Further, enhanced UVB radiation affected plasma immunoglobulin concentration. The results demonstrate that juvenile Atlantic salmon are not able to fully adapt to increased ambient UVB levels in long-term exposures, and the interference with immune system function suggests a negative effect of UVB on disease resistance in Atlantic salmon.

Effects of Montreal municipal sewage effluents on immune responses of juvenile female rainbow trout (Oncorhynchus mykiss).

The objective of this study was to examine the immunotoxicity of treated Montreal sewage effluents on juvenile female rainbow trout (Oncorhynchus mykiss). A comprehensive panel of immunological assays was used to evaluate the effects of exposure for 1 and 4 weeks to 1, 3, 10 and 20% sewage effluent. Phagocytic ingestion of fluorescent latex beads by head kidney macrophages and granulocytes was suppressed following 1-week of exposure, with the highest exposure concentration being the most suppressive. Phagocytic activity returned to control levels after 4 weeks of exposure. The cytotoxic activity of head kidney derived non-specific cytotoxic cells was enhanced after a 1-week exposure, especially at the lowest exposure concentration, and returned to control levels after 4 weeks of exposure. In vitro lymphocyte proliferation in response to LPS and ConA activation was not affected following sewage effluent exposure, but nonactivated, spontaneous proliferation of lymphocytes was suppressed in a dose-dependent manner after 4 weeks of exposure. Plasma lysozyme activity was elevated at lowest exposure concentration after 4 weeks. No changes were noted in either the blood leukocyte/erythrocyte ratio or in the proportion of circulating lymphocytes and thrombocytes. The proportion of circulating granulocytes increased following exposure for 4 weeks to the low effluent concentration. Plasma cortisol levels were not affected by effluent exposure suggesting that mechanisms other than stress influenced the observed immunomodulation. In summary, this study demonstrates that sewage effluent can alter the immune functions of rainbow trout.

Different sensitivity of carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) to the immunomodulatory effects of UVB irradiation.

In order to study the sensitivity of two fish species, carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss), to the immunomodulatory effects of ultraviolet B (UVB) radiation, the fish were exposed to a single UVB dose of 50, 250, 500 or 1,000 mJ cm(-2). These species represent different phylogenetic groups of fish, and they differ also in their behaviour inhabitating often dark and turbid (carp) or clear and transparent waters (salmonids). Immune responses were studied on day 1 post-irradiation. Unexposed fish, and fish exposed to radiation depleted of UV wavelengths served as controls. UVB irradiation markedly enhanced the blood respiratory burst and cytotoxic activity in carp, but in the head kidney these parameters were significantly suppressed. Rainbow trout respiratory burst was affected only after exposure with the highest dose of UVB. Lymphopenia and granulophilia were noted in both fish blood after exposure. This study indicates that UVB irradiation modulates immune functions in both fish species studied, and that rainbow trout is more tolerant than carp against UVB. Fish are clearly adapted to the environmental UVB levels prevailing in their usual living habitats, but are also a target of undesired effects of UVB on immune functions whenever exposed to increased radiation levels.

Effects of short- and long-term ultraviolet B irradiation on the immune system of the common carp (Cyprinus carpio).

Carp (Cyprinus carpio) were repeatedly exposed to 0, 60, 120 and 240 mJ/cm2 ultraviolet B (UVB) radiation three times in 1 week (short-term exposure) or 12 times in 4 weeks (long-term exposure). The effect of UVB on the functioning of the carp immune system was studied on day 2 after the final irradiation. After short-term UVB exposure, the whole-blood respiratory burst and cytotoxic activity were markedly enhanced, with parallel responses in both the number of circulating granulocytes and in the plasma cortisol concentration of the fish. These changes were not detectable after long-term exposure. The respiratory burst by head kidney granulocytes was suppressed dose dependently after both exposures, but cytotoxic activity was not affected. Exposure to UVB also modulated lymphocyte functions: nonstimulated and PHA-stimulated proliferation of head kidney lymphocytes in vitro was enhanced by both short-term and long-term exposure. LPS-stimulated proliferation was not affected by exposure nor was the number of immunoglobulin-secreting cells in the head kidney. In long-term exposure, the highest dose reduced the level of plasma IgM. This study indicates that UVB irradiation induces immunomodulation in the blood and head kidney of the carp and that the effects of short- and long-term exposure differ from each other. The results emphasize the potentially harmful impact of increased solar UVB radiation on fish immune functions.