Fusobacterium nucleatum lipopolysaccharide induces THP-1 cells to polarize towards M2 and produce a low concentration of IL-6

Abstract:

[Background] Fusobacterium nucleatum identified in many kinds of tumors is closely related to tumor initiation, poor prognosis, recurrence, and chemotherapy resistance in colorectal tumor. However, the mechanisms of F. nucleatum inducing inflammation and affecting immune cells such as macrophages in tumor microenvironment remain to be elucidated. [Objective] We explored the role and mechanism of F. nucleatum in the process of inducing chronic inflammation and cancer by comparing the monocyte polarization and inflammatory cytokine expression induced by Fusobacterium nucleatum-, Akkermansia muciniphila-, and Escherichia coli-derived lipopolysaccharides (LPSs). [Methods] After the treatment with A. muciniphila LPS, E. coli LPS, F. nucleatum LPS alone or combined with interferon-γ (IFN-γ), we observed the morphological changes of THP-1 and THP-1 (M0) cells. Further, we determined the mRNA levels of macrophage marker genes [including M0 (CD11B), M1 (CD40, CD86), and M2 (CD163, CD206)], TLR3, TLR4, IL-6, and IL-10 as well as the protein levels of IL-6, IL-10, and C-reactive protein. [Results] PAGE results showed that the LPSs from the three microbial species were significantly different in position and number of bands. F. nucleatum LPS possessed stronger activity of inducing adhesion of THP-1 cells. Meanwhile, the group treated with F. nucleatum LPS alone or in combination with IFN-γ had shorter pseudopodia and lower proportion of cells with pseudopodia and spindle-shaped cells (M1 cells) than the groups treated with A. muciniphila LPS and E. coli LPS. The LPSs from A. muciniphila, E. coli, and F. nucleatum up-regulated the mRNA level of CD40 by 5 011.0% (P<0.001), 6 048.9% (P<0.001), and 1 011.6% (P=0.009 4) and that of CD86 by 637.3% (P<0.001), 657.9% (P<0.001), and 194.1% (P>0.05), respectively. The LPSs down-regulated the mRNA level of CD163 by 39.5% (P=0.001 1), 53.7% (P<0.001), and 5.9% (P>0.05) and that of CD206 by 18.6% (P>0.05), 88.4% (P=0.005 5), and 24.8% (P>0.05), respectively. They down-regulated the mRNA level of TLR3 by 32.3% (P=0.044 7), 311.5% (P=0.001 9), and 9.6% (P>0.05), up-regulated that of IL-6 by 17 763.2% (P<0.001), 35 458.2% (P<0.001), and 1 123.6% (P>0.05), and up-regulated that of IL-10 by 729.3% (P<0.001), 1 223.3% (P<0.001), and 124.4% (P>0.05), respectively. The THP-1 cells treated with the LPSs of A. muciniphila, E. coli, and F. nucleatum alone produced IL-6 at 0.16 pg/mL, 6.17 pg/mL, and 0 pg/mL , and those treated with the LPSs in combination with IFN-γ produced IL-6 at 410.03 pg/mL, 1 334.40 pg/mL, and 46.20 pg/mL, respectively. [Conclusion] F. nucleatum LPS possessed a strong activity of recruiting monocytes and inducing them to polarize toward M2. It induced macrophages to produce a much lower amount of IL-6 than the LPSs of A. muciniphila and E. coli, which may play a role in triggering chronic inflammation and tumor immune response and escape. These findings suggest that studying the structure, activity, and mechanism of LPS from carcinogenic, immunomodulating or tumor therapy-associated bacteria will facilitate the elucidation of the role of these bacteria in chronic inflammation and tumorigenesis, which will provide new targets and strategies for the prevention and treatment of these diseases.

Key words:Fusobacterium nucleatum;lipopolysaccharide;THP-1;IL-6;chronic inflammation;monocyte