Studies Of The Effect On Immunoregulary Cells And The Related Mechanisms Induced By Sterigmatocystin

Sterigmatocystin (ST) is the carcinogenic metabolite producted by Aspergillus versicolor, Aspergillus nidulans etc. Contaminations of ST and it producing fungi are quite commonly seen in grains and animal diets all over the world. ST could be detected even in carpet dust from damp dwellings. Our previous study showed that both foodstuffs containing ST and oral administration of ST could induce adenocarcinoma of lung in NIH mice. ST could induce malignant transformation of human fetal gastric and lung cells in vitro. And studies both in vivo and in vitro showed that apart from its carcinogenic effects, ST could affect the proliferation, apoptosis and secretion of cytokines as well as the antigen presentation mechanisms of human and experimental animal immunocytes.As we all know that immune system plays a key role in the protection of human body against injuries of different environmental factors. Wide variety of etiological factors, especially biological factors, cause human diseases by weakening or destroying immune fuctions. Some extrinic factors could also affect carcinogenesis by its direct or indirect injury effects on immune mechnisms, especially by their injury effects on immune survillence. The impairment of immune system provides important foundation for the carcinogenesis processes. Currently, studies on the mechanism of ST induced damage to the immune system have centered on the direct immune injuries, such as those on proliferation, apoptosis and cytokine secretion of lymphocytes. Few studies involved in the immune regulatory cells. Therefore, it is of very important significance to explore the effect of ST on the immune regulatory cells so as to have a better understanding of the possible carcinogenic effects and other putative the biological effects of ST on human beings. Regulatory T cells (Tregs) are important subpopulation of T lymphocyte in immune system. Tregs are thought to be essential for maintaining tolerance to self-antigens recognised by autoreactive T cells that escape deletion in the thymus. An absence of functional Tregs has been associated with several autoimmune diseases. Tregs play important roles suppressing effective immune surveillance of carcinogen-induced tumours in intact animals. Naturally occurring Tregs specifcally express the transcription factor Foxp3 (forkhead box P3), a member of the fork-head/winged-helix family of transcription factors. Foxp3 is a master regulator of Treg development and function. Dendritic cell (DC) is an important antigen presenting cell (APC), which may interact with T, B and NK cells to promote their activation, differentiation and producing effects to induce or regulate primary or secondary immune response. Plasmacytoid dendritic cell (pDCs) is a member of DC family, and is of key importance in the development of chronic inflammation and autoimmune disease.As a downstream target of the T cell receptor and key protein in the Ca2+-dependent signal transduction pathway,Calcineurin (CaN) participates in the activation process of various lymphocytes. Cutaneous T cell-attracting chemokine 27 (Ccl27) is an important regulatory factor for the tumor-related T lymphocytes. The changes in the activity alteration of CaN and Ccl27 are thought to be closely associated with the development of various kind of skin diseases. Up to now, no studies on the possible effects of ST on Tregs, pDCs and CaN, Ccl27 expression have been seen.The aim of the present study is to further evaluate the putative effects of ST on the immunoregulary cells and to explore its possible mechanisms. Based on previous studies, we observed the effects of ST on the FoxP3+ regulatory T cells and plasmacytoid dendritic cells of peripheral blood, thymus, spleen as well as the skin tissue, and the expression of CaN and Ccl27 in the skin was also evaluated in mice.The effects of ST on the FoxP3+ regulatory T cells in human peripheral blood mononuclear cells and on MAPK signal transduction pathway were studied. The study includes three parts:1 Effects of sterigmatocystin on the immunoregulary cells in peripheral blood and immune organs of BALB/c miceObjective: To study the effects of the intraperitoneal injecting ST on the FoxP3+ regulatory T cells and plasmacytoid dendritic cells in peripheral blood mononuclear cell and immune organs in BALB/c mice.Methods: Male BALB/c mice were intraperitoneally injected with 3μg/kg, 30μg/kg, 300μg/kg and 3000μg/kg ST for 24 hours and the whole blood in peripheral vein was collected by eyeball enucleation. The murine peripheral blood mononuclear cells (MPBMCs) were isolated with Ficoll-Meglucamine Diatrizoate density gradient centrifugation. Thymus and spleen were taken after all the mice were sacrificed. The percentage of CD4+/CD8+ T cells and FoxP3+ T cells was determined using Epics-XLⅡflow cytometry with immunofluorescence labeling. The changes of FoxP3+ regulatory T cells and CD123+/BDCA2+ plasmacytoid dendritic cells number in thymus and spleen were studied with immunohistochemical staining. The expressions of FoxP3, CD123 and BDCA2 at protein and mRNA levels were measured by Western blot and RT-PCR.Results:1.1 Effect of ST on CD4+, CD8+ and FoxP3+ regulatory T lymphocyte in MPBMCsThe results of FCM showed that no significant difference was found in the percentage of CD4+ and CD8+ T cells in MPBMCs between all the ST groups with different dosage of ST in comparison with solvent control group. However, the percentage of FoxP3+ T cells was significant increased as the concentration of ST increases (r=0.862, n=5, P<0.01).RT-PCR analysis showed that FoxP3 was increased at mRNA level in MPBMCs(P<0.05), and there was a significant dose-effect correlation between ST dosage and the expression of FoxP3 at mRNA level.1.2 Effect of ST on FoxP3+ regulatory T lymphocyte in thymus and spleen in BALB/c mice The results of FCM showed that 24 hours after ST treatment, no significant differences were found in in the percentage of CD4+ T cells among control, solvent control and all ST treatment groups. However, in thymus, the percentage of CD8+ T cells was decreased in small dosage ST (3μg/kg) treatment group (P<0.05). While in spleen, the increases in percentage of CD4+ and CD8+ T cells was noted in small dosage ST (3 and 30μg/kg) treatment groups (P<0.05) but not in high dosage ST (300 and 3000μg/kg) treatment groups. Attentively, FCM results showed that the percentages of FoxP3+ T cells in all ST treatment groups were significantly increased both in thymus and spleen. A dose-effect correlation was found between ST dosage and the percentages of FoxP3+ T cells (P<0.05).In ST 30μg/kg, 300μg/kg and 3000μg/kg treatment groups, the immunohistochemical labelling index of FoxP3+ cells in thymus was obviously higher than that in solvent control group (P<0.05). And in spleen, FoxP3+ T cells in all ST treatment group were higher as compared with solvent controls (P<0.05). Moreover, within the dosage range from 0 to 3000μg/kg, there was a significant dose-effect correlation between ST dosage and the percentage of FoxP3+ T cells (thymus: r=0.831, P<0.01; spleen: r=0.873, P<0.01).The results of Western blotting showed that as compared with solvent control, the expressions of FoxP3 at protein level in all ST treatment groups were significantly increased. Within the ST dosage range from 3μg/kg to 3000μg/kg, positive correlation was found between the ST dosage and FoxP3 protein expression (thymus: r=0.658, P<0.01; spleen: r=0.621, P<0.01). The results of RT-PCR showed that FoxP3 was increased at mRNA level in murine thymus and spleen after different dosages of ST exposure for 24 hrs (P<0.05).1.3 Effect of ST on plasmacytoid dendritic cells in thymus and spleen of BALB/c miceIn comparison with solvent controls, the immunohistochemical labelling index of CD123+ cells in ST 30μg/kg, 300μg/kg, 3000μg/kg treatment groups were obviously lower in thymus and higher in spleen(P<0.05). And the immunohistochemical labelling index of BDCA2+ cells in thymus in 30μg/kg, 300μg/kg, 3000μg/kg ST treatment groups were obviously reduced, but in spleen, that in all ST groups were increased compared with solvent controls (P<0.05). Moreover, within the dosage range from 0 to 3000μg/kg, there was a significant dose-effect correlation between ST dosage and the percentage of CD123~+ and BDCA2+ cells.Western blotting results showed that, compared with solvent control group, a significant negative correlation between ST dosage and the expression of CD123 and BDCA2 at protein level in thymus could be found (CD123: r=-0.825, n=5, P<0.01; BDCA2: r=-0.831, n=5, P<0.01) within the dosage range from 3μg/L to 3000μg/kg. But in spleen, a significant positive correlation between ST dosage and the expression of CD123 and BDCA2 at protein level was seen (CD123: r=0.819, n=5, P<0.01; BDCA2: r=0.756, n=5, P<0.01).The results of RT-PCR suggested that CD123 was decreased at mRNA level in murine thymus and was increased in spleen in all ST treatment groups (P<0.01). Results from dosage-dependent studies indicated that there was a significant dose-effect correlation between ST dosage and the expression of CD123 mRMA both in thymus and spleen (thymus: r=-0.944, n=5, P<0.01; spleen: r=0.906, n=5, P<0.01).Thus,the results in this part suggestted that the effects of ST on pDCs be organ sprcific. The effects are totally different in central and peripheral lymphpoid oagans.2 Effects of sterigmatocystin on the expression of CaN and Ccl27 and FoxP3~+ regulatory T lymphocyte in the skin of BALB/c miceObjective: To evaluate the effects of single intraperitoneal administration of ST on expression of CaN and Ccl27 and the infiltration of FoxP3~+ regulatory T lymphocyte in the skin of BALB/c mice. Methods: The treatment of experimental animals was the same as in the first part. The full-thick skin specimen were obtained from the mice. Representative tissues specimens were fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin and sectioned. The changes in the number of CaN+, Ccl27+ and FoxP3~+ cells in skin tissues was studied with immunohistochemical staining method. Fresh skin tissues (100mg) for Western Blot was first homogenized in 500μL lysis buffer and then the total protein was extracted from the skin tissues and stored at -80℃. The expression of CaN, Ccl27 and FoxP3 protein was determined by Western blot. The expression of CaN, Ccl27 and FoxP3 at mRNA level was detected by semi-quantitative RT-PCR.Results:2.1 Effects of ST on the histopathological changes in the skin in BALB/c miceNo significant pathologial changes could be found in the skin in all the ST groups as compared with the controls.2.2 Effects of ST on the expression of CaN and Ccl27 in the skin tissues in BALB/c miceThe immunohistochemical labelling index of CaN+ cells of the skin in ST 30μg/kg, 300μg/kg and 3000μg/kg treatment groups were obviously higher than that in solvent control group (P<0.05). And within the dosage range from 0 to 3000μg/kg, the number of CaN+ cells was correlated with ST dosage (r=0.752, n=5, P<0.01). But the immunohistochemical labelling index of Ccl27+ cells in ST treatment groups were not different from that in solvent control group.The results of Western blot showed that as compared with solvent control, the expression of CaN at protein level was increased. A significant positive correlation could be found between ST dosage and the expression of CaN at protein level in the skin (r=0.931, n=5, P<0.01) within the dosage range from 3μg/kg to 3000μg/kg. But there was no difference of the expression of Ccl27 protein in ST treatment groups as compared with solvent control group.Similar to the results with immunohistochemical and Western blot, the results of RT-PCR showed that CaN was increased at mRNA level in murine skin after ST treatment for 24 h (P<0.01). And the expression of CaN mRMA in the skin had a significant dose-effect correlation with ST dosage (r=0.843, n=5, P<0.01). But no changes in Ccl27 mRNA expression could be found. 2.3 Effects of ST on FoxP3+ regulatory T lymphocyte in the skin tissues of BALB/c miceImmunohistochemical staining results showed that the positive labelling index of FoxP3+ cells in skin tissue of 300μg/kg and 3000μg/kg ST treatment groups were 3.30±0.675 and 4.70±0.949 respectively,which was obviously higher than that in solvent control group (1.40±0.966, P<0.05, P<0.05). And within the dosage range from 0 to 3000μg/kg, there was a significant dose-effect correlation between ST dosage and the percentage of FoxP3+ T cells (r=0.757, n=5, P<0.01).The results of Western blotting showed that, in comparison with solvent control, a significant dose-effect correlation could be found between ST dosage and the expression of FoxP3 at protein level in the skin (r=0.781, n=5, P<0.01) within the dosage range from 3μg/kg to 3000μg/kg.RT-PCR results showed that FoxP3 was increased at mRNA level in murine skin after exposure to different dosages of ST for 24 h (P<0.05). Results from concentration-dependent studies indicated that there was a significant positive correlation between ST dosage and the expression of FoxP3 mRNA (r=0.749, n=5, P<0.01).3 Effects of ST on FoxP3+ regulatory T lymphocyte in human peripheral blood mononuclear cell in vitro and the possible mechanismsObjective: To evaluate the effects of single treatment of ST on FoxP3+ regulatory T lymphocyte in human peripheral blood mononuclear cells in vitro and to explore the potential mechanisms.Methods:The human peripheral blood mononuclear cells (HPBMCs) were isolated with Ficoll-Meglucamine Diatrizoate density gradient centrifugation. After culture for 48 h, HPBMCs were harvestd, centrifuged and resuspended in 1640 medium supplemented with 10% FCS at the concentration of (1~2)×108 cells/L in culture flasks (8 ml). The medium of HPBMCs was replaced by new 1640 medium supplemented with 2% FCS 24 h later. Then the cells in ST groups were respectively treated with ST in different concentration of 100μg/L, 500μg/L, 1000μg/L and 2000μg/L, while their counterparts in solvent control and control group were incubated with DMSO and saline respectively. The cells were cultured for 24 h after treatment and harvested for detection. The percentage of CD4+/CD8+ T cells and FoxP3~+ T cells was determined using Epics-XLⅡflow cytometry with immunofluorescence labeling. The protein expression of FoxP3, JNK, ERK, p38 and the phosphorylation of JNK, ERK, p38 in HPBMCs treated with ST were determined by Western blot. The expression of FoxP3, JNK, ERK and p38 mRNA of HPBMCs cells was detected by RT-PCR.HPBMCs were harvestd 48 h after culture, centrifuged and resuspended in 1640 medium supplemented with 10% FCS at the concentration of (1~2)×108 cells/L in culture flasks (8 ml). HPBMCs were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L. The medium of HPBMCs cells was replaced by new 1640 medium supplemented with 2% FCS 24 h later. The cells of blocking agent groups were pretreated for 30 min with 1μM SP600125 (inhibitor of JNK), 50μM PD98059 (inhibitor of ERK), 0.5μM SB203580 (inhibitor of p38) and 1μM LY-294002 (inhibitor of PI3K) respectively. Then the cells in blocking agent +ST 1000μg/L group were treated with ST 1000μg/L, while these in solvent control and control groups were incubated with DMSO and saline respectively. Cells were harvested 24 h after ST treatment. The phosphorylation of JNK, ERK, p38 and the expression of FoxP3 protein in HPBMCs treated with ST were determined with Western blot. And the expression of FoxP3 mRNA of HPBMCs was detected by RT-PCR.Results:3.1 Effect of ST on CD4+, CD8+ and FoxP3~+ regulatory T lymphocyte in HPBMCs in vitroThe results of FCM showed that no differences were observed in the percentage of CD4+ and CD8+ T cells in MPBMCs between all the ST groups after exposure to different dosages of ST for 24 h and solvent control group. However, the percentage of FoxP3~+ T cells was significant increased in all ST treated groups and within ST dosage range from 0 to 2000μg/L, the percentage of FoxP3~+ T cells increased as the ST dosage increased (r=0.920, n=3, P<0.01).3.2 Effect of ST on the expression of FoxP3 protein in HPBMCs in vitroThe results of Western blot showed that as compared with solvent control, FoxP3 protein expressions were in all ST treatment groups. A significant positive correlation could be found between ST dosage and the expression of FoxP3 at protein level in the skin (r=0.868, n=3, P<0.01) within the dosage range from 0 to 2000μg/L.3.3 Effects of ST on the expression of FoxP3 mRNA in HPBMCs in vitroThe results of RT-PCR showed that the expression of FoxP3 mRNA in HPBMCs in all ST treatmen groups was increased compared with solvent control group (P<0.05), and there was a significant positive correlation between ST dosage and the expression of FoxP3 mRNA (r=0.793, n=3, P<0.01).3.4 Effects of ST on MAPK signal transduction pathway in HPBMCs3.4.1 Effects of ST on the expression and phosphorylation of JNK, ERK and p38The results of Western blot indicated that the expression of JNK, ERK and p38 protein in HPBMCs was not influenced by the ST treatment. No difference in the expression level of JNK, ERK and p38 was found between all the ST treatment groups and solvent control group (DMSO) (P>0.05). The phosphorylated JNK and ERK protein in ST treatment groups was significantly increased as compared with corresponding solvent control groups (p-JNK: r=0.831, n=3, P<0.01; p-ERK: r=0.687, n=3, P<0.01). A significant negative correlation between ST dosages and the phosphorylation of p38 was found (r=-0.661, n=3, P<0.01).The results of RT-PCR confirmed that no difference in the expression of JNK, ERK and p38 mRNA in HPBMCs was found between all the ST treatment groups and corresponding solvent control group (P>0.05).3.4.2 Effects of inhibitors on the expression of JNK, ERK and p38 induced by STThe results of Western blot showed that SP600125 and PD98059 pretreatment could block the increase in the phosphorylation of JNK and ERK induced by ST treatment n HPBMCs in vitro (P<0.05). The phosphorylation of p38 in HPBMCs could be inhibited by either ST 1000μg/L or SB203580 (0.5μM). And SB203580 (0.5μM) pretreatment could further decrease the the phosphorylation decrease of p38 by ST (P<0.05).3.5 Effects of MAPK signal transduction pathway on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by ST in vitro3.5.1 Effects of JNK signal transduction pathway activation on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by STWestern blot results showed that though the expression level of FoxP3 protein in SP600125+1000μg/L ST treated cells was significantly higher than that in solvant control group (P<0.05), but it was not different from that in 1000μg/L ST treatment cells (P>0.05).The findings of RT-PCR confirmed the same result for the expression of FoxP3 mRNA as for the expression of FoxP3 protein.Thus, the results in this study suggested that SP600125 had no effect on the increase of expression level of FoxP3 at protein and mRNA level induced by ST.3.5.2 Effects of ERK signal transduction pathway activation on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by STThe results of Western blot showed that the expression level of FoxP3 protein in PD98059+1000μg/L ST treatment group was significantly lower than that in 1000μg/L ST group (P<0.05). While there was no difference in the expression level of FoxP3 between PD98059+1000μg/L ST treatment group and solvant control group (P>0.05).RT-PCR results revealed that the expression level of FoxP3 mRNA in PD98059+ST treatment group also was significantly lower than that in ST group (P<0.05), and was no different from that in solvent control group.The results in this study indicated that the increase in the expression level of FoxP3 at protein and mRNA level induced by ST could be blocked by PD98059.3.5.3 Effects of p38 signal transduction pathway inhibition on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by STThe results of Western blot showed that SB203580 treatment could increase the expression level of FoxP3 protein compared with the solvant control group, and the combination treatment of SB203580 and ST could induce a significant increase of the FoxP3 protein expression as compared with both solvant control group and ST groups (P<0.05).The results of RT-PCR confirmed that the expression of FoxP3 mRNA in SB203580+ST treatment group was significantly increased as compared with that in solvent control group and ST group (P<0.05).The results in this study suggested that SB203580 could promote the increase in the expression of FoxP3 at protein and mRNA level induced by ST.Conclusions:1. Intraperitoneal administration of ST could significantly increase the percentage of FoxP3+ regulatory T cells and the expression of FoxP3+ at mRNA level in peripheral blood mononuclear cells in BALB/c mice.2. Intraperitoneal administration of ST could significantly increase the percentage of FoxP3+ regulatory T cells and the expression of FoxP3+ at protein and mRNA level in a dose-dependent pattern both in thymus and spleen of BALB/c mice.3. The effects of ST on pDCs are organ sprcific. The effect is totally different in central and peripheral lymphpoid oagan in BALB/c mice. Intraperitoneal administration of ST could significantly decrease the number of CD123+/BDCA2+ plasmacytoid dendritic cells in thymus, but increase that in spleen. The expression of CD123 and BDCA2 protein and CD123 mRNA was decreased in thymus while increased in spleen. 4. Intraperitoneal administration of ST could significantly increase the expression of CaN at protein and mRNA level in murine skin, but had no effect on that of Ccl27.5. Intraperitoneal administration of ST could significantly increase the infiltration of FoxP3+ regulatory T cells and the expression of FoxP3+ at protein and mRNA level in a dose-dependent pattern in murine skin.6. ST could significantly increase the percentage of FoxP3+ regulatory T cells and the expression of FoxP3+ at protein and mRNA level in HPBMCs.7. ST could activate JNK and ERK signal trusduction pathway, but inhibit p38 signal trusduction pathway in HPBMCs in vitro.8. JNK signal transduction pathway inhibitor SP600125 pretreatment had no effect on the increase of FoxP3 expression induced by ST. But ERK signal transduction pathway inhibitor PD98059 pretreatment could block the increase of FoxP3 expression induced by ST. And p38 signal transduction pathway inhibitor SB203580 had synergistic effect with ST on the increase of FoxP3 expression. ERK and p38 signal transduction pathway may be involved in FoxP3+ expression in HPBMCs in vitro.