<i>Fusobacterium nucleatum</i>-Induced Impairment of Autophagic Flux Enhances the Expression of Proinflammatory Cytokines via ROS in Caco-2 Cells

<div><p><i>Fusobacterium nucleatum</i> (<i>F</i>. <i>nucleatum</i>) plays a critical role in gastrointestinal inflammation. However, the exact mechanism by which <i>F</i>. <i>nucleatum</i> contributes to inflammation is unclear. In the present study, it was revealed that <i>F</i>. <i>nucleatum</i> could induce the production of proinflammatory cytokines (IL-8, IL-1β and TNF-α) and reactive oxygen species (ROS) in Caco-2 colorectal) adenocarcinoma cells. Furthermore, ROS scavengers (NAC or Tiron) could decrease the production of proinflammatory cytokines during <i>F</i>. <i>nucleatum</i> infection. In addition, we observed that autophagy is impaired in Caco-2 cells after <i>F</i>. <i>nucleatum</i> infection. The production of proinflammatory cytokines and ROS induced by <i>F</i>. <i>nucleatum</i> was enhanced with either autophagy pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or ATG12) in Caco-2 cells. Taken together, these results indicate that <i>F</i>. <i>nucleatum</i>-induced impairment of autophagic flux enhances the expression of proinflammatory cytokines via ROS in Caco-2 Cells.</p></div

Inhibition of autophagy enhances cytokines production and ROS generation induced by <i>F</i>. <i>nucleatum</i> infection.

<p>(A, B, and C) After pretreatment of 0.1% DMSO, 3-MA (2mM), Baf-A1 (10 nM) or Rapa (100 nM), Caco-2 cells were infected with <i>F</i>. <i>nucleatum</i> (MOI = 100:1) for 12 hrs. Supernatants of medium were assessed by ELISA for levels of IL-8, IL-1β and TNF-α. (D, E and F) Production of IL-8, IL-1β and TNF-α in Caco-2 cells transfected with siRNA specific for ATG5 or ATG12 (50 nM) for 24h and infected with <i>F</i>. <i>nucleatum</i> (MOI = 100) for 12 hrs, as assessed by ELISA. (G) siRNAs targeting ATG5 or ATG12 (100 nM each) transfected Caco-2 cells for 24 hrs, and the protein levels were assayed by Western blotting. (H and I) After pretreatment of 0.1% DMSO, 3-MA (2mM), Baf-A1 (10 nM) or Rapa (100 nM), or transfected with siRNA specific for ATG5 or ATG12 (50 nM) for 24h, Caco-2 cells were infected with <i>F</i>. <i>nucleatum</i> (MOI = 100:1) for 12 hrs. ROS generation was detected by DCFH-DA assay. The data shown are the means ±SEM of three experiments. *, <i>P</i><0.05.</p

TLR2/TLR4 regulate <i>F</i>. <i>nucleatum</i>-induced inflammatory cytokines through Tregs in vivo.

<p>(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 <i>F</i>. <i>nucleatum</i> for 1 week, as assessed by enzyme-linked immunosorbent assay (ELISA). (D) The number of <i>F</i>. <i>nucleatum</i> 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 <i>F</i>. <i>nucleatum</i> for 1 week and in the uninfected group was assessed by ELISA.</p

Schematic of the proposed mechanism of <i>Fusobacterium nucleatum</i>-induced impairment of autophagic flux enhancing the expression of proinflammatory cytokines via ROS in Caco-2 Cells (see text for details).

<p>Schematic of the proposed mechanism of <i>Fusobacterium nucleatum</i>-induced impairment of autophagic flux enhancing the expression of proinflammatory cytokines via ROS in Caco-2 Cells (see text for details).</p

TLR2/TLR4 are involved in the <i>F</i>. <i>nucleatum</i>-induced inflammatory signaling pathway.

<p>(A) Signaling pathways of differentially expressed genes in Caco-2 cells infected with <i>F</i>. <i>nucleatum</i>. 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 (<i>P</i> value) of these significant pathways. (B and C) Caco-2 cells or C57BL/6 mice were infected with <i>F</i>. <i>nucleatum</i> 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 <i>F</i>. <i>nucleatum</i> 16s rRNA gene copies in human clinical specimens was examined by Spearman correlation analysis and found to be positive (TLR4/<i>F</i>. <i>nucleatum</i>, R = 0.324, <i>P</i> = 0.12; TLR2/<i>F</i>. <i>nucleatum</i>, R = 0.618, <i>P</i> = 0.006).</p

LysoSensor Green DND-189 and DND-153 labeling of <i>E. chaffeensis</i>-infected DH82 cells.

<p>(A and D): The confocal images containing DAPI-stained nuclei and <i>E. chaffeensis</i> morulae (blue); (B): LysoSensor Green DND-153-labeled infected DH82 cells; (C): merged image of A and B; (E): LysoSensor Green DND-189-labeled infected DH82 cells (green); (F): merged image of D and E; (G): DAPI-stained uninfected DH82 cells; (H): LysoSensor probe-labeled uninfected DH82 cells; (I): merged image of G and H. Arrows indicate <i>E. chaffeensis</i> morulae. These results were confirmed in three independent labeling experiments.</p

TLR2/TLR4 signaling modulates <i>F</i>. <i>nucleatum</i>-induced inflammation in vivo.

<p>(A) Histological inflammation scores (H&E staining) of the gastric mucosa of C57BL/6 wild-type mice and TLR2^-/- and TLR4^-/- mice with <i>F</i>. <i>nucleatum</i> 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 <i>F</i>. <i>nucleatum</i> 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 <i>F</i>. <i>nucleatum</i> for 1 week, as assessed by enzyme-linked immunosorbent assay (ELISA). (F) The number of <i>F</i>. <i>nucleatum</i> 16s rRNA gene copies in the gastric mucosa of C57BL/6 wild mice and TLR2^-/- and TLR4^-/- mice based on qRT-PCR.</p

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

<div><p>Toll-like receptors (TLRs) 2 and 4 play critical roles in intestinal inflammation caused by <i>Fusobacterium nucleatum</i> (<i>F</i>. <i>nucleatum</i>) 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 <i>F</i>. <i>nucleatum</i>-induced inflammatory signaling pathway in Caco-2 cells, C57BL/6 mice and human clinical specimens. In TLR2^-/- and TLR4^-/- mice, <i>F</i>. <i>nucleatum</i> 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 <i>F</i>. <i>nucleatum</i>-induced inflammatory cytokines through Tregs in vivo.</p></div

<i>F</i>. <i>nucleatum</i> infection increased the ratio of regulatory T cells in vivo.

<p>(A) Sixteen C57BL/6 wild mice were divided into two groups (n = 8). After eight C57BL/6 wild-type mice were infected with <i>F</i>. <i>nucleatum</i> for 1 week, FoxP3 and CD4 expression was analyzed with FACS after fixation and permeabilization, and a gating control was used. The <i>P</i> 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.</p

Inflammation induced by <i>F</i>. <i>nucleatum</i> is dependent on ROS.

<p>(A) Caco-2 cells were infected by <i>F</i>. <i>nucleatum</i> for the indicated periods of time (3, 6, 12, 24 hours). ROS generation was detected by DCFH-DA assay. (B, C and D) Following pretreatment with 1 mM Tiron or 10 mM N-acetyl-cysteine (NAC) for 6 hours, Caco-2 cells were infected with <i>F</i>. <i>nucleatum</i> (MOI = 100:1) for 12 hours. Supernatants of medium was assessed by ELISA for levels of IL-8, IL-1β and TNF-α. The data are presented as the means ±SEM of at least 3 independent experiments. *, <i>P</i><0.05.</p