The Role Of Akkermansia Muciniphila On Regulating Macrophage Polarization And Suppressing Colorectal Carcinogenesis

Background and AimColorectal cancer（CRC）is one of the most common malignant tumors,which seriously endangers national health.Finding the characteristic events in the occurrence and development of CRC can provide new targets for the diagnosis and treatment of CRC.Recent studies have shown that the occurrence and development of CRC is closely related to the intestinal microbiota.Most studies focused on the role and mechanism of"harmful bacteria"such as Fusobacterium nucleatum and Bacteroides fragilis on the progression of CRC,but only a few studies reported the role of intestinal"good bacteria"in CRC.Akkermansia muciniphila（Akk）is a gram-negative anaerobic bacterium of the Verrucomicrobia phylum.Studies have reported that Akkermansia muciniphila can significantly inhibit the occurrence of colorectal tumors,but the molecular mechanism of Akkermansia muciniphila inhibit the colorectal tumor has not yet been elucidated.The intestinal microbiota is considered to be an important component of the host immune microenvironment for CRC.An in-depth analysis of the role and mechanism of Akkermansia muciniphila on the microenvironment of CRC is expected to provide important research ideas for the microbiota-host crosstalk.Materials and Methods1.The fecal Akkermansia muciniphila abundance of CRC in the GMrepo database was analyzed,and the fecal and tissue abundance of Akkermansia muciniphila of CRC patients collected from Sir Run Run Shaw Hospital of Zhejiang University were examined by q-PCR.Furthermore,we built up the Apc ^Min/+spontaneous adenoma model and subcutaneous tumor model on nude mice,detecting the effect of Akkermansia muciniphila on the tumor growth ability of Apc ^Min/+mice and tumor-bearing mice.2.Multi-flow cytometry tested the percentage of T cells and Myeloid cells in the colon tumor of Apc ^Min/+mice.The macrophage polarization was confirmed by high-throughput liquid protein chip and immunohistochemistry.The effect of Akkermansia muciniphila on the polarization of bone marrow-derived macrophages（BMDMs）and mouse macrophage Raw264.7 was detected by flow cytometry.The effect of macrophage on the proliferation of colon cancer cells was tested by CCK8.3.The NLPR3 inflammasome response of Akkermansia muciniphila on macrophage or tumor tissue were examined by western blot.Application of NLRP3inhibitor MCC950 or NLRP3 knockout mice(NLRP3^-/-),flow cytometry,CCK8 test,AOM/DSS model and subcutaneous growth explored the role of NLRP3 in the Akkermansia muciniphila-mediated macrophage polarization and tumor suppression in vivo and in vitro.4.The influence of Akkermansia muciniphila on the TLR2 and NF-κB pathways of macrophage or tumor tissue were detected by q-PCR and western blotting.Western blotting and flow cytometry analyzed the effect of TLR2 on NF-κB/NLRP3 pathways and its role in Akkermansia muciniphila-mediated macrophage polarization when using TLR2 inhibitor CPT22.5.The expression of i NOS and CD68⁺NLRP3⁺cells in colorectal cancer tissues with different Akkermansia muciniphila abundance were tested by immunohistochemistry and multi-immunofluorescence methods.The expression of NLRP3,TLR2 and their correlation in cancer tissues or adjacent tissues were examined by q-PCR.Results1.The fecal or tissue abundance of Akkermansia muciniphila in patients with CRC was significantly down-regulated compared to normal people.Besides,Akkermansia muciniphila can effectively inhibited the tumor progression in Apc ^Min/+mice and tumor-bearing nude mice.2.In Apc ^Min/+model,Akkermansia muciniphila significantly increased the proportion of M1-like TAM,while other immune cells such as M2-like TAM,myeloid-derived suppressor cells,dendritic cells,CD8⁺T cells and CD4⁺T cells had no difference.Furthermore,we found that Akkermansia muciniphila up-regulated M1macrophage-related cytokines and increased the level of i NOS in tumor tissues.Co-culturing BMDMs with Akkermansia muciniphila,or BMDMs with colon cancer cells proved that Akkermansia muciniphila promoted macrophage to M1 polarization in vitro,and M1 macrophage inhibited the proliferation of colon cancer cells.3.Akkermansia muciniphila up-regulated the protein levels of NLRP3,IL-1βprecursor and its cleavage protein IL-1β（p17）in vivo and in vitro.Inhibition or knockout of NLRP3 in vivo and in vitro significantly weakened Akkermansia muciniphila-mediated macrophage M1 polarization and macrophage inhibitory effect on CRC.4.Akkermansia muciniphila treatment induced the up-regulation of pattern recognition receptor TLR2 and the activation of NF-κB pathway.Inhibition of TLR2 in BMDMs and Raw264.7 cells abolished Akkermansia muciniphila-induced activation of NF-κB pathway,NLRP3 pathway or macrophage M1 polarization.5.In our CRC cohorts,Akkermansia muciniphila abundance had a positive correlation with the expression of i NOS and macrophage NLRP3.In our CRC cohorts and the TCGA database,the expression levels of NLRP3 or TLR2 in the cancer tissues were lower than that in their adjacent tissues.In addition,NLRP3 and TLR2 showed a positive correlation.Conclusions1.Akkermansia muciniphila suppressed the occurrence of colorectal cancer.2.Akkermansia muciniphila promotes M1-like TAM polarization in vivo and in vitro.3.TLR2/NF-κB/NLRP3 pathway is involved in the process of Akkermansia muciniphila-mediated macrophage polarization.