Campylobacter jejuni disrupts protective Toll-like receptor 9 signaling in colonic epithelial cells and increases the severity of dextran sulfate sodium-induced colitis in mice

Abstract

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation associated with a dysregulated immune response to commensal bacteria in susceptible individuals. The relapse of IBD may occur following an infection with Campylobacter jejuni. Apical epithelial Toll-like receptor 9 (TLR9) activation by bacterial DNA is reported to maintain colonic homeostasis. We investigated whether a prior C. jejuni infection disrupts epithelial TLR9 signaling and increases the severity of disease in a model of mild dextran sulfate sodium (DSS) colitis in mice. In a further attempt to identify mechanisms, T84 monolayers were treated with C. jejuni followed by a TLR9 agonist. Transepithelial resistance (TER) and dextran flux across confluent monolayers were monitored. Immunohistochemistry, Western blotting, and flow cytometry were used to examine TLR9 expression. Mice colonized by C. jejuni lacked any detectable pathology; however, in response to low levels of DSS, mice previously exposed to C. jejuni exhibited significantly reduced weight gain and increased occult blood and histological damage scores. Infected mice treated with DSS also demonstrated a significant reduction in levels of the anti-inflammatory cytokine interleukin-25. In vitro studies indicated that apical application of a TLR9 agonist enhances intestinal epithelial barrier function and that this response is lost in C. jejuni-infected monolayers. Furthermore, infected cells secreted significantly more CXCL8 following the basolateral application of a TLR9 agonist. Surface TLR9 expression was reduced in C. jejuni-infected monolayers subsequently exposed to a TLR9 agonist. In conclusion, infection by C. jejuni disrupts TLR9-induced reinforcement of the intestinal epithelial barrier, and colonization by C. jejuni increases the severity of mild DSS colitis.

Fig 1

C. jejuni infection increases clinical disease activity of mild DSS colitis. (A) Percent change in body weight in C57BL/6 mice. Three days after the start of DSS, infected mice exhibited a significant drop in weight compared to control mice, whereas mice treated with DSS or C. jejuni alone had weight gain similar to that of control mice. By 12 days of DSS treatment, infected mice had gained significantly less weight than all other groups (n = 7 or 8 mice/group; mean ± SEM). *, P < 0.05 compared to controls; σ, P < 0.05 compared to DSS-treated mice. (B) C. jejuni-infected mice showed an increased incidence of occult blood following DSS treatment compared to all other groups (n = 15 to 16 mice/group; mean ± SEM). *, P < 0.05.

Fig 2

C. jejuni infection increases the severity of histological inflammation in a model of mild DSS colitis. (A) Representative micrographs of H&E-stained colonic sections. Colons from control (upper left panel) and C. jejuni-treated (upper right panel) mice appeared microscopically similar. Control mice treated with low levels of DSS (lower left panel) displayed mild levels of inflammation. In contrast, infected mice treated with a low level of DSS exhibited extensive inflammation, as evidenced by increased mucosal thickening, inflammatory infiltrate, and ulceration. Photos were taken at the same magnification. (B) Histological damage scores in control and C. jejuni-, DSS-, and C. jejuni-plus-DSS-treated mice. Infected mice treated with DSS demonstrated a significantly increased histopathological score compared to control mice (n = 5 to 8 mice/group; mean ± SEM). *, P < 0.05.

Fig 3

Modulation of Th17 cytokine levels following DSS treatment in C. jejuni-infected mice. IL-25 (A) and IL-17 (B) levels were significantly reduced in colonic homogenates from C. jejuni-infected mice treated with DSS (n = 7 or 8 mice/group; mean ± SEM). ***, P < 0.0001; **, P < 0.01; *, P < 0.05.

Fig 4

Representative micrographs comparing TLR9 immunofluorescence results in the colons of sham- and C. jejuni-treated mice. Magnifications (×40) and exposure times are the same. TLR9 fluorescence is notably reduced in colonic epithelial cells following infection with C. jejuni (right panel) compared to sham-treated controls (left panel).

Fig 5

C. jejuni attenuates TLR9-induced increase in epithelial barrier function. (A) Transepithelial resistance of T84 monolayers was significantly increased in response to the administration of ODN 2006 for 24 h to the apical compartment of transwell units. Basolateral application of ODN 2006 failed to elicit a change in TER (n = 18; mean ± SEM). ***, P < 0.0001. (B) The apical-to-basolateral flux of a FITC-dextran probe was significantly lower in cells treated with apical ODN 2006 (n = 15 to 16/group; mean ± SEM). ***, P < 0.0001; **, P < 0.001. (C and D) T84 cells pretreated with C. jejuni for 24 h did not exhibit a significant change in TER (C) or apical-to-basolateral flux of a FITC-dextran probe following the administration of ODN 2006 for an additional 24 h to the apical or basolateral chambers (D) (n = 15 to 18/group; mean ± SEM).

Fig 6

C. jejuni infection modulates TLR9 protein expression in epithelial cell monolayers. (A) Representative micrographs of TLR9 immunofluorescence, demonstrating the redistribution of TLR9 away from the cell membrane in C. jejuni-infected monolayers (lower left panel). Infected monolayers treated with ODN 2006 (lower right panel) exhibit further redistribution. (B) Loss of TLR9 protein levels as determined by immunoblotting. Densitometry data are expressed as percent GAPDH (n = 9/group; mean ± SEM). ***, P < 0.0001; **, P < 00.1.

Fig 7

Surface TLR9 expression is lost in C. jejuni-infected intestinal epithelial cells following ODN 2006 treatment. Live T84 cells were labeled with an anti-hTLR9 antibody and analyzed by flow cytometry for surface detection. (A) Representative fluorescence-activated cell sorter histograms comparing surface expression of TLR9 in control (upper left panel) and ODN 2006 (upper right panel)-, C. jejuni (lower left panel)-, and C. jejuni-plus-ODN 2006-treated intestinal epithelial cells (lower right panel). The green histogram represents the level of surface TLR9 expression. The blue-filled histogram represents the unlabeled cells. (B) The surface expression of TLR9 is significantly reduced following ODN 2006 treatment in C. jejuni-infected intestinal epithelial cells (n = 6/group; mean ± SEM). *, P < 0.05.

Fig 8

C. jejuni infection potentiates ODN 2006-induced CXCL8 secretion. Control and C. jejuni-infected T84 monolayers were treated apically or basolaterally with ODN 2006 for 24 h, and CXCL8 secretion was measured by ELISA. Basolateral stimulation with ODN 2006 induced CXCL8 secretion from control cells. Cells previously infected with C. jejuni secreted significantly more CXCL8 in response to basolateral stimulation with ODN 2006 (n = 24/group; mean ± SEM). *, P < 0.05.

Fig 9

TLR9-induced increases in TER are independent of NF-κB, MAPK, and PI3K signaling. Pretreatment of T84 cell monolayers with an NF-κB (Bay 11-7085; 10 μmol/liter) (A), MAPK (apigenin; 20 μmol/liter) (B), or PI3K (LY294002; 10 μmol/liter) (C) inhibitor for 1 h did not attenuate the increase in TER induced by the administration of ODN 2006 to the apical compartment (n = 12 to 16/group; mean ± SEM). *, P < 0.05.