Recombinant Helicobacter pylori neutrophil-activating protein fused with maltose-binding protein (rMBP-NAP), a potential TLR2 ligand, was reported to possess immunomodulatory effects on in situ tumors in our previous study. In the present work, we attempt to elucidate the effect of rMBP-NAP at the local immune modulation in B16-F10-induced metastatic lung cancer. Our results demonstrated that growth of B16-F10 melanoma metastases in the lung was significantly arrested after rMBP-NAP treatment, along with marked reduction in metastatic lung nodules and significant increase in survival. The treatment induced both local and systemic immune responses, which were associated with higher influx of CD4/CD8 T cells and drove toward Th1-like and cytotoxic immune environment. Moreover, rMBP-NAP also showed significant anti-angiogenic activity by reducing vascularization in lung tumor sections. rMBP-NAP could induce antitumor immunity through activating Th1 cells and producing pro-inflammatory cytokines, which are responsible for the effective cytotoxic immune response against cancer progression. Our findings indicate that rMBP-NAP might be a novel antitumor therapeutic strategy.
Asthma is a chronic inflammatory airway disease. It was prevalently perceived that Th2 cells played the crucial role in asthma pathogenesis, which has been identified as the important target for anti-asthma therapy. The soluble IL-4 receptor (sIL-4R), which is the decoy receptor for Th2 cytokine IL-4, has been reported to be effective in treating asthma in phase I/II clinical trail. To develop more efficacious anti-asthma agent, we attempt to test whether the Helicobacter pylori neutrophil-activating protein (HP-NAP), a novel TLR2 agonist, would enhance the efficacy of sIL-4R in anti-asthma therapy. In our work, we constructed a pcDNA3.1-sIL-4R-NAP plasmid, named PSN, encoding fusion protein of murine sIL-4R and HP-NAP. PSN significantly inhibited airway inflammation, decreased the serum OVA-specific IgE levels and remodeled the Th1/Th2 balance. Notably, PSN is more effective on anti-asthma therapy comparing with plasmid only expressing sIL-4R.
Summary
During eukaryotic cell mitosis, the nuclear envelope disintegrates and transcription factors are dissociated from condensed chromosomes. Here, we describe a protocol to study centrosomal translocation of nuclear receptor RXRα. We detail procedures for HeLa cell synchronization followed by immunofluorescence, in situ proximity ligation assay, and centrosome isolation. This protocol can be used to identify other transcription factors associated with the centrosome or other subcellular structures during mitotic progression.
For complete details on the use and execution of this protocol, please refer to
Xie et al. (2020)
The pro-inflammatory and immunomodulatory properties of Helicobacter pylori neutrophil activating protein (Hp-NAP) not only make it play an important role in disease pathogenesis, but also make it a potential candidate for applications, including vaccine and drug development. Our previous work demonstrated that the recombinant Hp-NAP fused with the maltose-binding protein of Escherichia coli (rMBP-NAP) exert an important role in regulating the differentiation of Th1 cells. As a potential TLR2 ligand, it was reported to possess the ability to induce systemic antitumor immunity in murine hepatoma H22 and sarcoma S180 tumor models. To further understand the antitumor and immunomodulatory effect of rMBP-NAP, we elucidate the effect and mechanism of rMBP-NAP at the local immune response modulation in established mouse B16-F10 melanoma pulmonary metastasis model. Our results demonstrated that metastatic lung tumor growth was significantly arrested after rMBP-NAP treatment, along with marked reduction in the number of lung nodules and significant increase in survival. Flow cytometry immunophenotyping and Quantitative RT-PCR analyses demonstrated that rMBP-NAP could induce both local and systemic immune responses, which associated with higher influx of CD3+CD4+T cells, CD3+CD8+T cells and higher secretion of interferon (IFN)-γ and interleukin (IL)-27 cytokines. The intraperitoneal administration of rMBP-NAP in mice promoted infiltration of lymphocyte in the lungs and reduced the production of several proinflammatory cytokines, such as IL-6 and transforming growth factor beta (TGF) -β, indicating an anti-inflammatory effect in local area. By comparing with control mice, mRNA expression of chemokines also revealed that rMBP-NAP treatment substantially decreased the expression of CCL2 and CCL20 in tumor tissues, which are central to recruit myeloid-derived suppressor cell (MDSC) and regulatory T cells (Treg) to tumor microenviroment and form immunosuppression. Moreover, we assess the expression of vascular marker CD34 in pulmonary section using immunohistochemical method, the results showed the vascularization was apparently inhibited in rMBP-NAP treated tumor bearing mouse. In conclusion, rMBP-NAP could induce the activation of effective Th1/Tc1 cells and the production of relevant inflammatory cytokines and chemokines, which are responsible for enhencing T cell immunity and reversing immunosuppression against metastatic cancer progression. Our findings strongly indicate that rMBP-NAP treatment might be a novel therapeutic approach against metastatic melanoma, and rMBP-NAP might be a potential template for the development of agents that could be used as immunomodulatory adjuvant in the therapy against melanoma.
Note: This abstract was not presented at the meeting.
Citation Format: Ting Wang, Cong Ding, Zhenyu Ji, Xin Liu, Mingxuan Du, Qiaozhen Kang. Recombinant protein MBP-NAP restricts tumor progression by triggering T-cell immunity in mouse metastatic melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 166. doi:10.1158/1538-7445.AM2017-166
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