TLR2/TLR4 activation induces Tregs and suppresses intestinal inflammation caused by Fusobacterium nucleatum in vivo

Abstract

Toll-like receptors (TLRs) 2 and 4 play critical roles in intestinal inflammation caused by Fusobacterium nucleatum (F. nucleatum) infection, but the role of TLR2/TLR4 in regulation of proinflammatory cytokines remains unknown. In this study, through microarray analysis and qRT-PCR, we showed that TLR2/TLR4 are involved in the F. nucleatum-induced inflammatory signaling pathway in Caco-2 cells, C57BL/6 mice and human clinical specimens. In TLR2-/- and TLR4-/- mice, F. nucleatum infection resulted in increased colonization of the bacteria and production of the proinflammatory cytokines IL-8, IL-1β and TNF-α. In addition, the ratio of Foxp3+ CD4+ T cells in the total CD4+ T cells in TLR2-/- and TLR4-/- mice was less than that in wild-type mice, and the ratio in hybrid mice was more than that in knockout mice, which suggested that TLR2/TLR4 mediated the number of Tregs. Furthermore, it was observed that inflammatory cytokine levels were reduced in TLR2-/- mice after Treg transfer. Thus, these data indicate that TLR2/TLR4 regulate F. nucleatum-induced inflammatory cytokines through Tregs in vivo.

Fig 1. TLR2/TLR4 are involved in the F. nucleatum-induced inflammatory signaling pathway.

(A) Signaling pathways of differentially expressed genes in Caco-2 cells infected with F. nucleatum. Pathway analysis was predominantly based on the KEGG database. An AP-value of <0.05 and an FDR of <0.05 in the two-sided Fisher’s exact test were considered statistically significant. The vertical axis represents the pathway category, and the horizontal axis represents the log 10 (P value) of these significant pathways. (B and C) Caco-2 cells or C57BL/6 mice were infected with F. nucleatum for 24 h or 1 week. The mRNA levels of TLR1, TLR2, TLR4, TLR5 and TLR6 were determined by qRT-PCR. (D) The correlation between TLR2/TLR4 and F. nucleatum 16s rRNA gene copies in human clinical specimens was examined by Spearman correlation analysis and found to be positive (TLR4/F. nucleatum, R = 0.324, P = 0.12; TLR2/F. nucleatum, R = 0.618, P = 0.006).

Fig 2. TLR2/TLR4 signaling modulates F. nucleatum-induced inflammation in vivo.

(A) Histological inflammation scores (H&E staining) of the gastric mucosa of C57BL/6 wild-type mice and TLR2^-/- and TLR4^-/- mice with F. nucleatum infection for 1 week. The intensity of staining is shown in the right graph, and the data are expressed as the mean±SEM. (B) The body weight of C57BL/6 wild-type mice and TLR2^-/- and TLR4^-/- mice after F. nucleatum infection. (C, D and E) The production of IL-8, IL-1β and TNF-α in C57BL/6 wild mice and TLR2^-/- and TLR4^-/- mice infected with F. nucleatum for 1 week, as assessed by enzyme-linked immunosorbent assay (ELISA). (F) The number of F. nucleatum 16s rRNA gene copies in the gastric mucosa of C57BL/6 wild mice and TLR2^-/- and TLR4^-/- mice based on qRT-PCR.

Fig 3. F. nucleatum infection increased the ratio of regulatory T cells in vivo.

(A) Sixteen C57BL/6 wild mice were divided into two groups (n = 8). After eight C57BL/6 wild-type mice were infected with F. nucleatum for 1 week, FoxP3 and CD4 expression was analyzed with FACS after fixation and permeabilization, and a gating control was used. The P value (Mann–Whitney) is indicated. (B) Detection of Foxp3⁺ CD4⁺ T cells in total CD4⁺ T cells in wild-type, TLR2^-/- and TLR4^-/- mice. (C) Detection of Foxp3⁺ CD4⁺ T cells in total CD4⁺ T cells in wild-type, hybrid, TLR2^-/- and TLR4^-/- mice.

Fig 4. TLR2/TLR4 regulate F. nucleatum-induced inflammatory cytokines through Tregs in vivo.

(A, B and C) Production of IL-8, IL-1β and TNF-α in C57BL/6 wild-type mice and in hybrid, TLR2^-/- and TLR4^-/- mice infected with F. nucleatum for 1 week, as assessed by enzyme-linked immunosorbent assay (ELISA). (D) The number of F. nucleatum 16s rRNA gene copies in the gastric mucosa of C57BL/6 wild-type mice and in hybrid, TLR2^-/- and TLR4^-/- mice determined by qRT-PCR. (E) After transfer of Tregs from wild-type mice to TLR2^-/- mice, the production of IL-8, IL-1β and TNF-α in TLR2^-/- mice infected with F. nucleatum for 1 week and in the uninfected group was assessed by ELISA.