Fusobacterium Nucleatum Promotes M2 Polarization Of Macrophages In The Microenvironment Of Colorectal Tumors Via A TLR4/IL-6/STAT3/c-MYC Cascade

Aims:Fusobacterium nucleatum（Fn）has been shown to promote colorectal cancer（CRC）development by inhibiting host antitumor immunity.However,the impact of Fn infection on macrophages polarization and subsequent intestinal tumor formation,and the underlying molecular pathways has not been investigated.Methods:（1）We used hematoxylin-eosin staining（HE）to classify the tissue types of collecting samples.（2）We measured Fn abundance in CRC tissues and intestinal tumor tissues in C57BL/6-Apc^min/+mice with fluorescence in situ hybridization（FISH）.（3）We used immunofluorescence staining to investigate eight immune cell markers（CD3,CD4,CD8,CD83,NE,CD68,CD86 and CD206）in colorectal cancer tissues.After that,we used multiple immunofluorescence to detect the co-localiztion of immune cells markers（CD3⁺,CD4⁺,CD8⁺,NE,CD83⁺and CD68⁺）and IL-6,as well as two types of macrophages（CD86⁺and CD206⁺）and c-MYC in 8 human CRC tissues with Fn infection.（4）We compared IL-6 and STAT3 protein abundance in Fn-positive and Fn-negative CRCs through immunohistochemistry（IHC）.（5）The murine peritoneal macrophages RAW264.7 cells were treated with four kinds of conditions,namely clinical strains Fn（F01）,Fn（F01）+TAK-242,Fn（ATCC 10953）and non-pathogenic E.coli,and each condition was observed for2h and 6h,respectively.TAK-242 was Toll-like receptor 4（TLR4）blockers,we treated RAW264.7 cells in advance 1h and then Fn（F01）was used to infect cells.Then we determined the cell density of M1 and M2 macrophages by immunofluorescence staining of CD86⁺or CD206⁺,and examined the co-localization of M1 and M2 macrophages with c-MYC.（6）The macrophages RAW264.7 cells were treated with different conditions of Fn（F01）,Fn（F01）+TAK-242,Fn（ATCC10953）and E.coli for 2h and 6h,then total RNA was extracted and reversely transcribed into c DNA.After that Quantitative real-time PCR PCR（Q-PCR）was used to detect the m RNA levels of IL-6,IL-12,TNF-α,IL-10,TGF-βand GAPDH.（7）These Apc^Min/+mice were treated with three different interventions for 8 weeks.After the model was successfully established,tumors in the colon and small intestine were counted and tumor volume was calculated.The numbers of various immune cells in the intestinal tumor tissues of the three groups and that of CD86⁺M1 macrophages and CD206⁺M2 macrophages co-localized with c-MYC were detected by the tissue immunofluorescence assay.The three different interventions are:Fn（F01）intragastric administration daily;FAB daily intragastric administration;Fn（F01）daily intragastric administration and TAK-242 intraperitoneal injection.Results:（1）Fn were detected in 69.0%of CRCs,29 cases were positive,13 cases were negative.（2）We observed CD68⁺macrophages were the most predominantly increased immune cell populations in Fn-positive CRCs（P<0.001）.（3）In Fn-positive CRCs,the density of M2 macrophages were significantly higher than M1 macrophages（P=0.001）.（4）We observed CD206⁺M2 macrophages were significantly increased in Fn（ATCC10953）and Fn（F01）challenged macrophages compared to E.coli（ATCC25922）challenged macrophages at both 2 and 6 hours（all P values<0.05）.Moreover,the m RNA levels of M2markers（IL-10,TGF-β）were determined by real-time quantitative PCR（Q-PCR）were significantly increased in Fn（F01）challenged macrophages（IL-10:P values<0.05,TGF-β:P=0.003,P=0.005）。（5）The CD206⁺M2 macrophages were significantly decreased in TAK-242pretreatment macrophages compared to Fn（F01）challenged macrophages（P<0.001）.Moreover,m RNA levels of M2 markers（IL-10 and TGF-β）were dramatically reduced in TAK-242 pretreatment group compared with Fn（F01）group（P<0.05）.（6）The Fn-fed mice had higher number of tumors（P=0.029）as well as larger intestinal tumor volume on average（P=0.027）compared to control group.（7）The density of CD68⁺macrophages in the microenvironment of intestinal tumors was significantly higher in Fn-fed mice compared to control group（P=0.002）and CD206⁺M2 macrophages were significantly higher in the microenvironment of intestinal tumors of Fn-fed mice than in control group（P<0.05）.（8）Expression of IL-6 and STAT3 was significantly higher in Fn-positive CRCs than that in Fn-negative CRCs（all P values<0.05）.（9）The frequency of immunofluorescence co-staining for macrophages（CD68⁺）and IL-6 was significantly higher than that for other immune cells（CD3⁺,CD4⁺,CD8⁺,NE and CD83⁺）and IL-6 in Fn-positive CRCs（all P values<0.05）.（10）The m RNA of IL-6 was expressed at a significantly higher level in Fn（F01）challenged macrophages compared to Fn（ATCC10953）and E.coli challenged macrophages at 2 and 6 hours（all P value<0.05）.Pretreatment with TAK-242 led to a significant decrease of IL-6 m RNA abundance compared to Fn（F01）treatment group（P<0.05）.（11）The frequency of co-staining for CD206⁺macrophages and c-MYC protein was significantly higher than that for CD86⁺macrophages and c-MYC in human CRCs with Fn infection（P<0.01）.（12）The frequency of co-staining for CD206⁺macrophages and c-MYC in Fn（F01）challenged cells was significantly higher than that in Fn（ATCC10953）and E.coli challenged cells.TAK-242 pretreatment significantly decreased the frequency of co-staining for CD206⁺macrophages and c-MYC compared to Fn（F01）challenged cells（P<0.05）.Conclusions:（1）Fn-positive CRC was predominantly infiltrated by macrophages,with M2macrophages more than M1 macrophages.（2）Fn infection RAW264.7 cells can promote its polarization to M2 macrophages.（3）Fn infection Apc^Min/+mice can promote both its number and volume ofintestinal tumors to increase,and contributetheir macrophages to M2 macrophage polarization.（4）Fn induced macrophages in the gut of Apc^Min/+mice to polarize M2 macrophages by activating TLR4 signaling.（5）IL-6 in Fn-positive CRC is mainly secreted by macrophages.（6）Fn infection CRC tissue can increase the expression of IL-6and STAT3.（7）The c-MYC is a specific signature of M2 macrophages activation.（8）Fn infection increased M2 polarization of macrophages in vitro and in vivo,enhanced colorectal tumor growth,which is likely to be associated with activated TLR4/IL-6/STAT3/c-MYC signaling.