Soybean glycinin- and β-conglycinin-induced intestinal immune responses in a murine model of allergy

Glycinin and β-conglycinin are major soybean allergens involved in food hypersensitivity. The study was aimed to investigate the soybean protein-induced intestinal immune responses and its possible mechanism. Balb/c mice were sensitised intragastrically with glycinin and β-conglycinin without an adjuvant for five weeks. Results showed that the sensitised mice displayed diarrhoea symptoms and jejunal morphological changes, including decreased villous height and thickened crypt depth. Both the histamine and immunoglobulin A levels in jejunum were increased in soybean allergen-sensitised mice. Moreover, the number of IgA+B cells and CD4+T cells in the jejunal lamina propria of sensitised mice were significantly increased. The expression levels of interleukin-4 and interferon-γ were increased, whereas the levels of IL-10 and transforming growth factor-β (TGF-β) were decreased in the jejunum mucosa. In conclusion, glycinin and β-conglycinin induce a mixed Th1/Th2 immune response. The suppression of IL-10 and TGF-β may play important roles in the development of intestinal allergic reactions.     

Thymic stromal lymphopoietin‐induced interleukin‐17A is involved in the development of IgE‐mediated atopic dermatitis‐like skin lesions in mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with elevated levels of allergen-specific IgE. Although thymic stromal lymphopoietin (TSLP) and interleukin-17A (IL-17A) have been considered as important factors in allergic diseases, their relationships in AD have not been fully defined. Here, we show the contribution of TSLP-induced IL-17A responses to IgE-mediated AD-like skin lesions. BALB/c mice passively sensitized by intraperitoneal injections of ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were challenged with OVA applied to the skin six times. Treatment with anti-TSLP mAb during the second to sixth challenges inhibited IgE-mediated AD-like skin lesions and IL-17A production in lymph nodes. Furthermore, the increased number of IL-17A-producing CD4⁺ and γδ T cells in lymph nodes and neutrophilic inflammation in the skin were reduced by anti-TSLP mAb. These findings prompted us to examine the roles of IL-17A. Treatment with anti-IL-17A mAb suppressed the AD-like skin lesions and neutrophilic inflammation; anti-Gr-1 mAb also inhibited them. Furthermore, treatment with CXCR2 antagonist reduced the AD-like skin lesions and neutrophilic inflammation accompanied by the reduction of IL-17A production; the increased CXCR2 expression in the epidermal cells was suppressed by anti-TSLP mAb. Meanwhile, these treatments, except for anti-Gr-1 mAb, inhibited the increased mast cell accumulation in the skin. Collectively, the mechanism of IgE mediating IL-17A-producing CD4⁺ and γδ T cells through TSLP by repeated antigen challenges is involved in AD-like skin lesions associated with skin inflammation, such as neutrophil and mast cell accumulation; TSLP may regulate CXCR2 signalling-induced IL-17A production.     

The association between phthalate exposure and atopic dermatitis with a discussion of phthalate induced secretion of interleukin-1β and thymic stromal lymphopoietin

Phthalate diesters are widely used as emollients in plastic and cosmetics as well as in food packaging and perfumes, potentially leading to prolonged and repeated dermal, oral and airborne exposure. We here review published articles that have evaluated the putative role of phthalate diesters in the pathogenesis of atopic dermatitis and discuss possible pathogenic pathways. A literature search resulted in 563 articles in Embase and 263 articles in Pubmed. After identification of relevant articles based on screening of titles, abstracts and reference lists, a total of 39 articles were selected and included. While no clear association has been shown between systemic phthalate levels and atopic dermatitis in human studies, animal data suggests that phthalates may worsen dermatitis and in vitro data suggests that interleukin-4 could be upregulated. Moreover, both loss-of-function mutations in the filaggrin gene and atopic dermatitis have been associated with elevated systemic phthalate levels. There is a need for prospective studies to clarify the possible pathogenic role of phthalate diesters in atopic dermatitis and the associated health risk, especially with the general trend towards barrier restoration with emollients in infants at risk of developing atopic dermatitis. In summary, we conclude that the results from published studies are controversial and inconclusive.     

Protein tyrosine phosphatase conjugated with a novel transdermal delivery peptide,astrotactin 1–derived peptide recombinant protein tyrosine phosphatase (AP-rPTP), alleviates both atopic dermatitis–like and psoriasis-like dermatitis

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The effect of TGF-beta1 on immune responses of naïve versus memory CD4+ Th1/Th2 T cells

The role of TGF-beta1 in the regulation of T cell responses has been perplexing, possibly because it is dependent on the type of T cell being regulated and its cytokine microenvironment. In the present study, we demonstrate that TGF-beta1 has a profound inhibitory effect on naive CD4+ T cell undergoing differentiation under defined neutral, Th1 and Th2 priming conditions. In addition, we show that if CD4+ T cells are primed in the presence of TGF-beta1, they exhibit reduced secondary anti-CD3/anti-CD28-induced and antigen-specific immune responses (even when TGF-beta is absent during the secondary response), which is not due to reduced expression of co-stimulatory molecules or to inadequate IL-2 production. Finally, with respect to the effect of TGF-beta on fully differentiated antigen-specific memory CD4+ T cells, we demonstrate that while antigen-specific activation and cytokine secretion by memory Th1 T cells is inhibited by TGF-beta1, such inhibition is associated with partial down-regulation of IL-12 receptor beta2 chain expression. In contrast, memory Th2 T cells are not subject to TGF-beta1 -mediated suppression. In summary, these studies reveal that TGF-beta1 is a powerful negative regulator of the primary immune response of CD4+ T cells, but only Th1 T cells are subject to such regulation after the memory stage of T cell differentiation has been reached. Thus, these studies define the potential regulatory role of TGF-beta1 in Th1 and Th2 T cell-mediated autoimmunity.     

Blockade of TSP1-dependent TGF-β activity reduces renal injury and proteinuria in a murine model of diabetic nephropathy

Transforming growth factor-β (TGF-β) is key in the pathogenesis of diabetic nephropathy. Thrombospondin 1 (TSP1) expression is increased in diabetes, and TSP1 regulates latent TGF-β activation in vitro and in diabetic animal models. Herein, we investigate the effect of blockade of TSP1-dependent TGF-β activation on progression of renal disease in a mouse model of type 1 diabetes (C57BL/6J-Ins2(Akita)) as a targeted treatment for diabetic nephropathy. Akita and control C57BL/6 mice who underwent uninephrectomy received 15 weeks of thrice-weekly i.p. treatment with 3 or 30 mg/kg LSKL peptide, control SLLK peptide, or saline. The effects of systemic LSKL peptide on dermal wound healing was assessed in type 2 diabetic mice (db/db). Proteinuria (urinary albumin level and albumin/creatinine ratio) was significantly improved in Akita mice treated with 30 mg/kg LSKL peptide. LSKL treatment reduced urinary TGF-β activity and renal phospho-Smad2/3 levels and improved markers of tubulointerstitial injury (fibronectin) and podocytes (nephrin). However, LSKL did not alter glomerulosclerosis or glomerular structure. LSKL did not increase tumor incidence or inflammation or impair diabetic wound healing. These data suggest that selective targeting of excessive TGF-β activity through blockade of TSP1-dependent TGF-β activation represents a therapeutic strategy for treating diabetic nephropathy that preserves the homeostatic functions of TGF-β.     

Protective effects of N-acetylcysteine on myocardial injury induced by hindlimb ischaemia–reperfusion: a histological study in a rat model

This study evaluated the effects of N-acetylcysteine as a scavenger of radical oxygen species on myocardial injury as a remote organ after skeletal muscle ischaemia–reperfusion. Twenty male Wistar rats were allocated randomly into two experimental groups: ischaemia–reperfusion and ischaemia–reperfusion?+?N-acetylcysteine. All animals underwent 2 h of ischaemia by occlusion of the femoral artery followed by 24 h of reperfusion. Rats treated with N-acetylcysteine were given an intravenous dose of 150 mg/kg, immediately before reperfusion. After the reperfusion period, animals were euthanized and hearts harvested for histopathological analysis under light microscopy. In the ischaemia–reperfusion group, tissues showed histological changes with interstitial oedema, neutrophil infiltration and adhesion of neutrophils to the endothelium, haemorrhage and coagulative necrosis. Histopathologically, there was a significant difference (P?N-acetylcysteine significantly decreased myocardial injury induced by skeletal muscle ischaemia–reperfusion according to our histological findings.     

Filaggrin null mutations are associated with atopic dermatitis and elevated levels of IgE in the Japanese population: a family and case–control study

Filaggrin (FLG) plays an important role in the barrier function of the skin. Several loss-of-function mutations in the FLG gene have been identified in patients with ichthyosis vulgaris, and these null mutations are associated with atopic dermatitis (AD) development. In this study, we examined tag single nucleotide polymorphisms (tSNPs) and null mutations in FLG for possible associations with AD and atopic phenotypes in a Japanese population. Transmission disequilibrium test of 105 AD families showed that the null allele of the S2554X variant of FLG tended to be overtransmitted to AD-affected offspring; however, the P value did not reach statistical significance. In a case–control comparison of 376 AD cases and 923 nonallergic controls, the null allele of S2554X was significantly associated with AD (P = 0.0012), and the association was strengthened in subjects with AD alone (P = 0.000024). We found that 3321delA and S2554X were also associated with elevated levels of immunoglobulin E (IgE). Combined null mutation carriers were observed more in AD patients and in subjects with high IgE than in control subjects. The combined P value for the family and case–control data was significant for the S2554X and combined null mutations. Our data further support the importance of FLG in AD development.     

Inhibitory effect of 1α-hydroxyvitamin D3 on N-nitrosobis(2-oxopropyl)amine-induced cholangiocarcinogenesis in Syrian hamsters

Sixty-three male 5-week-old Syrian hamsters received the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) s.c. in 5 weekly injections (the first, 70 mg/kg body, and the remaining, 20mg/kg each). The hamsters that received BOP were given intragastric administration of 0.2 ml of medium chain triglyceride (MCT) with or without 0.04 μg of 1α-hydroxyvitamin D3 [1α(OH)D3] through a feeding tube for 12 weeks. Thus, 3 groups were assigned:Group 1;BOP alone (n＝20), Group 2;BOP＋MCT (n＝18) and Group 3;BOP＋1α(OH)D3 (n＝25). The mean body weight of Group 3 was lower than those of Groups 1 and 2 at the end of the experiment (p＜0.001,Tukey-Kramer HSD test). At the end of week 12, all surviving hamsters were put to sleep. The incidences of liver tumors were 80%, 72% and 32% in Groups 1, 2 and 3, respectively. The incidence of tumors in Group 3 was significantly lower than in Group 1 and Group 2 (p＜0.05, χ2-test). All tumors were cholangiocarcinoma. These results indicated that BOP-induced cholangiocarcinogenesis was suppressed by the supplemental administration of 1α(OH)D3.     

Hypoxia,hypoxia-inducible factor-1α and vascular endothelial growth factor in a murine model of Schistosoma mansoni infection

Schistosomiasis mansoni is a chronic parasitic disease where much of the symptomatology is attributed to granuloma formation, an immunopathological reaction against Schistosoma eggs. To more clearly understand the immunopathology of schistosomiasis, the tissue microenvironment generated by S. mansoni infected mice was investigated. Using the hypoxia marker pimonidazole, we provide immunohistochemical evidence that hypoxia occurred in inflammatory cells infiltrated around the eggs and cells surrounding granulomas in the liver, intestine, spleen and lungs of infected mice. Hypoxia-inducible factor-1α (HIF-1α) was mainly expressed in inflammatory cells surrounding the eggs and in hepatocytes surrounding cellular and fibrocellular granulomas in infected mouse liver. HIF-1α expression was also verified in granulomas in the other tissues tested (intestine, spleen and lungs). Vascular endothelial growth factor (VEGF) expression was observed in the extracellular space surrounding inflammatory cells in liver granuloma. The VEGF expression pattern verified in infected mouse liver was very similar to that observed in the other tissues tested. A strong positive correlation occurred between pimonidazole binding and HIF-1α and VEGF expression in the tissues tested, except for lung. This work is the first evidence that infection by a helminth parasite, S. mansoni, produces a hypoxic tissue microenvironment and induces HIF-1α and VEGF expression.     

Concomitant suppression of TH2 and TH17 cell responses in allergic asthma by targeting retinoic acid receptor–related orphan receptor γt

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Effects of DNA polymerase kappa and mismatch repair on dose–responses of chromosome aberrations induced by three oxidative genotoxins in human cells

Genotoxic carcinogens are regulated under the policy that there is no threshold or safe dose. It has been pointed out, however, that self-defense mechanisms, such as detoxification, DNA repair, and error-free translesion synthesis, may protect chromosome DNA from genotoxic insults, thereby constituting practical threshold. In this study, we examined dose responses of chromosome aberrations induced by three oxidative genotoxins, that is, hydrogen peroxide (H₂O₂), menadione and paraquat, with or without DNA polymerase kappa (Polκ) activities and mismatch repair capacities in human cells. Polκ is involved in translesion synthesis across DNA damage and mismatch repair is responsible for correction of base–base mismatch in DNA. Polκ activity of the cells was inactivated either by point mutations in the catalytically essential amino acids (catalytically dead or CD) or by deletion of the POLK gene (knockout or KO). In the absence of mismatch repair, frequencies of chromosome aberrations induced by H₂O₂ and menadione were not significantly different among CD, KO, and the wild type (WT) cells. In the presence of mismatch repair, however, cytotoxicity and clastogenicity were enhanced and Polκ modulated the sensitivity of the cells. No-observed-genotoxic-effect-levels (NOGELs) for H₂O₂ and menadione were CD = KO < WT cells. In contrast, the sensitivities of the cells to paraquat were not significantly affected by the status of mismatch repair or Polκ activity. The results suggest that mismatch repair and Polκ coordinately modulate NOGELs for the clastogenicity of H₂O₂ and menadione and also that DNA lesion(s) responsible for paraquat-induced chromosome aberrations are different from those induced by H₂O₂ and menadione. Environ. Mol. Mutagen. 61:193–199, 2020. © 2019 Wiley Periodicals, Inc.     

T cells demonstrate a Th1-biased response to native β2-glycoprotein I in a murine model of anti-phospholipid antibody induction

Anti-phospholipid syndrome (APS) is an autoimmune disorder characterized by the presence of autoantibody (AAb) to phospholipid (PL)-binding proteins, such as β2-glycoprotein I (β2GPI), and clinical manifestations including thrombosis and/or recurrent pregnancy loss. β2GPI-reactive T cells are clearly implicated in the generation of these AAb, but the mechanism responsible for their activation remains unclear. We hypothesized that immunization of mice with human β2GPI, in the context of a potent innate immune activator lipopolysaccharide (LPS), would generate not only high titers of anti-PL AAb, but also a strong β2GPI-specific T cell response. Healthy, nonautoimmune C57BL/6 mice were immunized repeatedly with human β2GPI in the presence of LPS. High titers of anti-PL to β2GPI appeared after the second immunization, with T cell reactivity to β2GPI detectable only after the fourth immunization. Splenic T cells from these mice proliferated in response to native β2GPI, alone or bound to anionic PL. These T cells produced IL-2 and IFN-γ, but not IL-4 or IL-10, indicating a Th1 bias of the β2GPI-specific response. These findings suggest that T cells responsive to β2GPI may become activated in APS patients by exposure to their cognate Ag in the context of innate immune activation and a pro-inflammatory environment.     

The molecular mechanism of dexamethasone-mediated effect on the blood–brain tumor barrier permeability in a rat brain tumor model

This study was performed to determine whether dexamethasone (DEX) had an effect on calcium-activated potassium channels (K_Ca channels) and Occludin protein in blood–brain tumor barrier (BTB). Using a rat brain glioma model, we found that the expression of K_Ca channels protein and Occludin protein was significantly increased in brain tumor tissue after DEX treatment for 3 days. Compared with non-DEX-treated animals, Evans Blue levels were greatly attenuated in DEX-treated animals. These effects were significantly reversed by the glucocorticoid receptor antagonist RU38486. In addition, DEX treatment enhanced the density of I_KCa in the rat brain microvascular endothelial cells (RBMECs) in vitro BTB. All of these results strongly suggest that DEX could be involved in the regulation of both transcellular and paracellular pathway.     

Antileukemic effect of Kalpaamruthaa,a modified Siddha preparation,in BCR–ABL + cell line induced chronic myeloid leukemic mouse model

Kalpaamruthaa is a modified Siddha (herbal) drug prepared in our laboratory. It has been proven to have therapeutic effect against arthritis, mammary cancer, and liver cancer. To evaluate the antileukemic effect of the drug, Kalpaamruthaa (KA), in chronic myeloid leukemia-induced mice, leukemia was induced in BALB/c mice by tail vein injection of BCR–ABL ⁺12B1 murine leukemia cell line. Leukemia-induced animals were treated with KA at a dosage of 200 mg/kg b.wt. dissolved in 0.5 ml of olive oil for 14 days, daily by gastric intubation. Imatinib mesylate was used as the reference drug. The drug KA significantly decreased the levels of marker enzymes and lipid peroxides and increased the levels of antioxidant enzymes. The bone marrow and histopathological abnormalities were also found to be normalized after treatment with the drug, KA. No significant adverse effect was observed in sole drug-treated group of rats. The present study suggests that KA, by means of its antioxidant and free radical quenching effect, has definite anti cancer effect in leukemic mice.     

Prevention and treatment of allergic inflammation by an Fcγ-Der f2 fusion protein in a murine model of dust mite-induced asthma

The immunoglobulin E (IgE) high-affinity receptor FcεRI expressed on mast cells and basophils plays a critical role in triggering allergic disease. The co-aggregation of the FcεRI and FcγRIIb receptors is inhibitory to FcεRI signaling and holds great potential for the treatment of IgE-mediated allergies. In China, Dermatophagoides farinae is a common anaphylaxis trigger. Therefore, in this study, the FcγRIIb-mediated immunomodulating activity of recombinant Fcγ-Der f2 fusion protein was tested in a Der f2-allergic murine model. Following the treatment, bronchoalveolar lavage fluid (BALF) was collected to measure the expression of several Th1/Th2-type cytokines (IL-5, TNF-α, IL-12p70, IL-4, IL-10, IFN-γ and IL-18) and histamine, while blood was used to detect the specific IgE and IgG-types anti-Der f2 antibodies, for measurement. In contrast to the saline-treated allergic mice, the levels of Der f2-specific IgE, cytokines and histamine were lowered in the Fcγ-Der f2-treated allergic mice, in addition to the rare inflammatory cell infiltration in the airways and blood vessels revealed by histopathological examination. The recombinant Fcγ-Der f2 protein was demonstrated to function as an effective immunotherapeutic agent, suggesting that chimeric human Fcγ-allergen proteins could be used in the development of antigen-specific immunotherapy for human allergic diseases.     

Development and progression of immobilization-induced skin fibrosis through overexpression of transforming growth factor-ß1 and hypoxic conditions in a rat knee joint contracture model

Purpose: The purpose of this study was to investigate the pathology and mechanism of immobilization-induced skin fibrosis in a rat joint contracture model. Methods: Rats were randomly divided into control and immobilization groups. In the immobilization groups, knee joints of the rats were immobilized for 1, 2, and 4 weeks. After each immobilization, skin was dissected. To assess fibrosis in the skin, the thickness and area of adipocytes and connective tissue fibers were measured. Myofibroblasts were analyzed by immunohistochemistry by using anti-α-SMA as a marker. Gene expression levels of type I and III collagen, TGF-ß1, and HIF-1α were measured by using RT-PCR. Results: One week after immobilization, there was a marked increase in the area of connective tissue fibers in the immobilization group. Type I and type III collagen were significantly increased with prolonged immobilization. Higher numbers of α-SMA-positive cells were noted in the immobilized group at 2 and 4 weeks after immobilization. The expression level of TGF-β1 mRNA in the immobilization group increased after one week of immobilization. In contrast, the expression level of HIF1-α mRNA increased after 2 weeks of immobilization, and a greater increase was seen at 4 weeks after immobilization. Conclusions: These results suggest that immobilization induces skin fibrosis with accumulation of types I and III collagen. These fibrotic changes may be evoked by upregulation of TGF-β1 after one week of immobilization. Additionally, upregulation of HIF-1α may relate to skin fibrosis by accelerating the differentiation of fibroblasts to myofibroblasts starting at 2 weeks after immobilization.