| Hepatocellular carcinoma is the most common and deadliest form of primary liver tumors with limited treatment options. Nowadays, immunotherapy becomes a hot spot in liver cancer studies such as vaccine or immune cells related treatment.Lmdd-MPFG(LM) is a recombinant attenuated Listeria vaccine constructed in our previous study, which has potency to induce strong and specific antitumor cellular immunity to HCC. As the most powerful antigen-presenting cells, Dendritic cells(DCs) play an essential role in Lm infection. However, the Lm vaccine utilizes DCs to enhance antitumor immunity and the probable mechanisms have not been described. Here, we prefer to explore the function of DC in LM infection and clear the possible immune regulatory mechanisms in tumor inhibition, so we can open up a new way for tumor immunotherapy.In this study, we first observed the maturation of dendritic cells after the LM-MPFG vaccine stimulation in vitro. Immature DCs were stimulated by LPS, wild type LM(WT), Lmdd-MPFG(LM), and heat-killed LM or WT at MOI 20, respectively. DC function was examined by flow cytometry after stimulation and the cytokines were tested by ELISA. Also T cells specific proliferation was measured to understand the significance of the LM infection and our findings demonstrated Lmdd-MPFG could activate BMDCs in vitro and induce T cell responses.To understand the pathways, TLRs and NLRs related expressions were analyzed by real-time PCR and western blot. The related PRRs including NOD1, NLRP3 and TLR4 were important for DCs maturation after LM infection via NF-kB and Caspase-1 signaling pathway. To further confirm the effect on NLRP3 expression and the interactions between the NLRP3, NOD1/2 and TLR4 signaling pathways in the LM-induced DC-activation process, we collected immature DCs and subjected them to different treatments(control, MDP, LPS+MSU, MSU, MDP+MSU, SiNLRP3). After stimulation by corresponding agonists, DC functional phenotypes were detected by flow cytometry. Further, the expression of cytokines in the supernatants of each group was mesured by ELISA. Finally, we examined the protein expression of these receptors and the signaling pathway in cells of each group by western blot analysis. These data confirmed the essential function of NLRP3 and its crosstalk with NOD1/NOD2 and TLR4 in LM-induced DC presentation.Further, the role of DC in tumor microenvironment was investigated after LM immunization in tumor-bearing mice. Tumor sizes were measured and T cells differentiations in tumors were detected by flow cytometry. We found dendritic cells are required for LM infection in the spleen and promoted LM mediated antitumor effect in vivo. Also in DC adjuvant therapeutic mice, tumor suppression was accompanied by the most significant enhanced cytotoxic T lymphocyte response and an inducing CD4+T cell subsets differentiation especially the Th17 cells was presented. In addition, functional receptors between LM-MPFG and DC were investigated. As a result, we considered that the synergistic effect of TLR4 and NLRP3 or NOD1 signaling pathway may be a crucial component in LM-induced DC activation. At last, functional defects of dendritic cells were also showed in HCC patients.Collectively, these results suggest that LM-MPFG vaccine presents a potentially feasible strategy for prevention of HCC. Dendritic cells enhanced the tumor suppression effect after LM vaccine immunization. And the interaction of TLR4, NLRP3 and NOD1 is showed relevance in LM-induced DC maturation, which provides a probable immune regulatory mechanism in LM vaccine-related tumor immunotherapy. |
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