Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease

Abstract

Initiation and perpetuation of the inflammatory intestinal responses in inflammatory bowel disease (IBD) may result from an exaggerated host defense reaction of the intestinal epithelium to endogenous lumenal bacterial flora. Intestinal epithelial cell lines constitutively express several functional Toll-like receptors (TLRs) which appear to be key regulators of the innate response system. The aim of this study was to characterize the expression pattern of TLR2, TLR3, TLR4, and TLR5 in primary intestinal epithelial cells from patients with IBD. Small intestinal and colonic biopsy specimens were collected from patients with IBD (Crohn's disease [CD], ulcerative colitis [UC]) and controls. Non-IBD specimens were assessed by immunofluorescence histochemistry using polyclonal antibodies specific for TLR2, TLR3, TLR4, and TLR5. Primary intestinal epithelial cells (IEC) of normal mucosa constitutively expressed TLR3 and TLR5, while TLR2 and TLR4 were only barely detectable. In active IBD, the expression of TLR3 and TLR4 was differentially modulated in the intestinal epithelium. TLR3 was significantly downregulated in IEC in active CD but not in UC. In contrast, TLR4 was strongly upregulated in both UC and CD. TLR2 and TLR5 expression remained unchanged in IBD. These data suggest that IBD may be associated with distinctive changes in selective TLR expression in the intestinal epithelium, implying that alterations in the innate response system may contribute to the pathogenesis of these disorders.

FIG. 1

The specificity of newly generated anti-TLR antisera is confirmed using Western blotting against peptides. The human colon cancer cell line T84 was used as a presumed positive control for TLR protein expression.

FIG. 2

TLR2 expression is minimally detectable in IECs and remains unchanged in active IBD. (A) Normal, nondiseased mucosa. (C) UC, inactive. (B and D) CD, active colon involved. (Olympus IX70; original magnification, ×40; standardized exposure time, 1.039 s; full image area, gain 16; gamma adjust, 1.20; noise filter, no background subtraction.)

FIG. 3

TLR3 is constitutively expressed in the IECs of healthy controls and UC patients but significantly decreased in the IECs of CD patients. Shown are normal, nondiseased mucosa of the terminal ileum (A) and colon (B); inactive (C) and active (D) UC cells; and active, noninvolved CD colon cells (E) and active, involved CD colon cells (F). White arrows indicate scattered lamina propria mononuclear cells. Olympus AX70; original magnification, ×40; exposure times: 8 s (A), 10 s (B, C, and F), 9 s (D), and 16 s (E); Elitechrome Kodak Select series ISO100, Olympus exposure control unit PM-30, Superfluorescence 30 (exposure adjust setting, auto 2).

FIG. 4

The fluorescence intensity of TLR3 staining is decreased on average by 55 or 43% in CD samples compared to healthy controls or UC patients, respectively. Average automatic exposure times of representative samples (n = 7 per disease subgroup) were plotted as an indirect measurement of the fluorescence intensity of TLR3. t test (two tailed, heteroscedastic) results show the following: ∗, CD/Normal, P < 0.01; #, CD/UC, P < 0.03; +, UC/Normal, P > 0.7.

FIG. 5

TLR4 expression is minimal in the IECs of healthy controls but significantly increased in the IECs of both UC and CD. Shown are normal, nondiseased mucosae of the terminal ileum (A) and colon (B); inactive (C) and active (D) UC cells; and active, noninvolved CD terminal ileum cells (E) and active, involved CD colon cells (F). (Olympus IX70; original magnification, ×40; standardized exposure time, 1.039 s; full image area, gain 16; gamma adjust, 1.20; noise filter, no background subtraction.)

FIG. 6

Constitutive TLR5 is predominantly expressed on surface IECs in healthy controls and remains unchanged in IBD. Shown are an overview (A) and detail (B) of normal, nondiseased mucosa of the colon; active, noninvolved CD colon cells (C); and active UC cells (D). White arrows indicate surface epithelial staining. Olympus AX70; original magnifications, ×40 (B and D) and ×10 (A and C); exposure times: 4 s (A), 3 s (B), and 5 s (C and D); Elitechrome Kodak Select series ISO400, Olympus exposure control unit PM-30, Superfluorescence 30 (exposure adjust setting, auto 2).