| Pidotimod (Pido) is a synthetic immunomodulator with orally activity. It has been widely used in clinical research because of its ability to promote both non-specific immune response and the specific immune response. However, little is known about the mechanism of regulator function of pidotimod. Macrophages are a kind of immune effector cells in immune system and of great importance. Previous studies have demonstrated that macrophaged could sense series of stimuli from both endogenous and exogenous environment, and respond with corresponding phenotypic plasticity. The question is proposed whether pidotimod could induce the polarization of macrophage. Therefore, the aim of this research was to investigate the effectiveness of pidotimod on the polarization of macrophages, by determining the contribution of pidotimod on polarization and functions of mouse bone marrow-derived macrophages (BMDMs) in vitro, defining the use of pidotimod in mice OVA-induced asthma model, and comparing the activity of DSS-induced inflammatory bowel disease in response to M2macrophage and pidotimod-treated M2macrophage adoptive transport. The major results were as following:1. In vitro contribution of pidotimod on bone marrow-derived macrophages polarization and functionThis experiment was carried to test the effect of pidotimod on the polarization and function of BMDMs. There is no cytotoxicity of pidotimod within1-200μg/ml to BMDMs after culturing12h or24h. The results showed that0.1,1or10μg/ml pidotimod had no influence on LPS/IFN-γ-induced M1macrophage polarization. While interestingly, co-stimulation of pidotimod with IL-4significantly increased the expression of M2marker gene Argl, Fizzl, Yml and MR (p<0.01) in M2macrophages, indicating that pidotimod facilitated macrophage polarization into the M2type. And1μg/ml concentration of pidotimod was chosen to do the following research. Compared with M2macrophages, co-stimulation1μg/ml pidotimod and IL-4on macrophages for12h or24h, the expression of Argl was2.23-or2.21-fold higher (p<0.01), Fizzl expression was significantly up-regulated after6h treatment (p<0.01), Ym1expression was increased after24h (p<0.001), and12h later, the expression of MR was dramatically enhanced(p<0.001). Additionally, cell surface expression of mannose receptor was dramatically enhanced using fluorescence activated cell sorter (FACS) analysis after treated macrophage with pidotimod and IL-4for24h (p<0.001). Furthermore, the supernatant of pidotimod-treated M2macrophage could increase the migration (p<0.05) and enhance the wound closure rate (p<0.05) of MLE-12cells. Together, these data demonstrated that compared with IL-4-induced M2macrophages polarization, pidotimod could facilitate the M2macrophage polarization and its functions.2. Oral treatment with pidotimod in OVA-induced asthma miceAsthma is a kind of chronic airway inflammatory diseases, which is caused by dysregulated Th2-type immune responses. It has been reported that Th2cytokines could induce the polarization of M2macrophage and enhance the expression of M2marker genes, which contribute to the airway inflammatory and remodeling responses during asthma. The results showed that:(1) When the asthma mice were treated with pidotimod, the number of eosinophils was significantly increased compared with OVA group (p<0.05); and the total number of inflammatory cells and eosinophils in BALF were significantly improved when compared with Dex group (p<0.05, p<0.01).(2) When asthma mice intragastric administration of pidotimod, serum IgE antibody levels were significantly higher than Control group (p<0.001), OVA group (p<0.001) and Dex group (p<0.001). IL-4level in BALF from Pido group was significantly lower than the OVA group (p<0.01); while it had no difference compared with the Control group and Dex group (p>0.05), it still showed an increasing trend and indicated that pidotimod could not protect mice from asthma; there was no different between the IL-4level in fed the mice in Dex group with different concentrations Pido and Dex group. In Pido group, IL-5level in BALF were significantly improved p<0.001) compared with the Control group, OVA group and Dex group, while different concentrations of Pido were given to Dex mice, no difference of IL-5level in each group of mice with Dex group in BALF(p>0.05). IL-13level was dramaticly higher than the Control group (p<0.01) and Dex group (p<0.05), and compared with the OVA group, there was no difference (p>0.05).(3) Structural disruption and hypertrophy of the airway epithelial cells, and eosnophils infiltration was observed in Pido group mice, and inflammation score slightly higher than the OVA mice (p>0.05), while compared with the Dex group, it were significantly improved (p<0.001). A lot of goblet cells were observed in respiratory epithelium from the mice in Pido group, it was much more than that from the OVA group (p>0.05).(4) In Pido group, positive Fizzl stained particles in airway epithelium were significantly higher than Dex group (p<0.01), and there were positive colored particles in other lung organizations except the airway epithelium part compared with the OVA group (p<0.05). In Pido group, positive Argl stained particles in airway epithelium and other tissue in lung were observed, while the PAS scores was similar to the OVA group (p>0.05). These findings revealed that pidotimod could facilitate M2macrophage polarization and furtherly exacerbate OVA-induce asthma inflammation.3. In vivo adoptive transport with M2polarization macrophages in DSS-induced inflammatory bowel disease (IBD) mice modelPrevious researches have shown that M2-polarized macrophage might be protective in murine models of intestinal bowel inflammation. This experiment was conducted to compare the development of IBD disease, the cytokines level in sera and intestinal morphology, after adoptive transport with M2polarization macrophages and pidotimod-treated M2macrophages in DSS-induced IBD mice. The results showed that:(1) Significant weight loss was developed in DSS group mice compared with M2macrophages and pidotimod-treat M2macrophages adoptive transfer group, and at the10th day of experiment, weight loss in Pido-treated M2group was significantly slower than M2group (p<0.05). The disease activity index (DAI) of M2and Pido-treated M2group were both dramatically lower than DSS group (p<0.001).(2) Pro-inflammatory cytokine IEN-γ level in M2and Pido-treated M2group was dramatically inhibited relative to DSS group, and anti-inflammatory cytokine TGF-β1levels in pido-treated M2macrophages adoptive transfer group were significantly higher than the Control group (p<0.05), the DSS group (p<0.001), and M2group (p>0.05).(3) In pido-treated M2macrophages adoptive transfer group, either the structure disruption of intestinal villi and mucosa glands, or inflammatory cell infiltration in mucosa and submucosa were observed in the colonic section from pido-treated M2macrophages adoptive transfer group. The same result was found in M2group. And the histological scores of two adoptive transfer groups were significantly lower than DSS group. The above tests indicatied that pidotimod-treated M2macrophage could attenuate DSS-induced IBD and protect mice from inflammation response and intestinal structure disruption.All the data provided that pidotimod promoted IL-4-induced M2macrophage polarization and enhanced the M2macrophages’function of cell migration and tissue repair, and this result was testified by the OVA-induced asthma and DSS-induced IBD models in mice. |
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