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. 2007 Dec;117(12):3909-21.
doi: 10.1172/JCI33084.

Deletion of TLR5 results in spontaneous colitis in mice

Matam Vijay-Kumar  1 , Catherine J Sanders, Rebekah T Taylor, Amrita Kumar, Jesse D Aitken, Shanthi V Sitaraman, Andrew S Neish, Satoshi Uematsu, Shizuo Akira, Ifor R Williams, Andrew T Gewirtz
Affiliations

Deletion of TLR5 results in spontaneous colitis in mice

Matam Vijay-Kumar et al. J Clin Invest. 2007 Dec.

Abstract

Activation of TLRs by bacterial products results in rapid activation of genes encoding products designed to protect the host from perturbing microbes. In the intestine, which is colonized by a large and diverse population of commensal bacteria, TLR signaling may not function in a simple on/off mode. Here, we show that the flagellin receptor TLR5 has an essential and nonredundant role in protecting the gut from enteric microbes. Mice lacking TLR5 (TLR5KO mice) developed spontaneous colitis, as assessed by well-defined clinical, serologic, and histopathologic indicators of this disorder. Compared with WT littermates, TLR5KO mice that had not yet developed robust colitis exhibited decreased intestinal expression of TLR5-regulated host defense genes despite having an increased bacterial burden in the colon. In contrast, such TLR5KO mice displayed markedly increased colonic expression of hematopoietic-derived proinflammatory cytokines, suggesting that elevated levels of bacterial products may result in activation of other TLRs that drive colitis in TLR5KO mice. In accordance, deletion of TLR4 rescued the colitis of TLR5KO mice in that mice lacking both TLR4 and TLR5 also had elevated bacterial loads in the colon but lacked immunological, histopathological, and clinical evidence of colitis. That an engineered innate immune deficiency ultimately results in spontaneous intestinal inflammation supports the notion that an innate immune deficiency might underlie some instances of inflammatory bowel disease.

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Figures

Figure 1
Figure 1. TLR5KO mice develop spontaneous colitis.
(A) Representative rectal prolapse exhibited by about 10% of TLR5KO (T5KO) mice. (B) Representative gross appearance of cecum/colon of TLR5KO mice and WT littermates. (C) Comparison of organ weights from TLR5KO mice and WT littermates. (D) H&E-stained sections of cecum and proximal colon of TLR5KO mice and WT littermates. Note the extensive immune cell infiltrates, focal crypt epithelial destruction, edema, and epithelial hyperplasia in TLR5KO mice. (E) Immunofluorescent micrographs showing neutrophils stained with anti-GR1 (in red) and counterstained with DAPI to show all nuclei in blue. Scale bars: 100 μM. *P < 0.05.
Figure 2
Figure 2. SAA levels mark severity of colitis in TLR5KO mice.
Eight- to twelve-week-old TLR5KO mice and WT littermates were euthanized and parameters assayed. (A) SAA: open triangles represent mice with rectal prolapse at time of serum collection. SAA levels were then used to stratify mice for analysis in B–E. T5KO-H indicates SAA >100 μg/ml; T5KO-L indicates SAA< 50 μg/ml. (B) Immunoblot showing serum lipocalin 2. (C) Occult blood in feces (D) Weights of cecum, colon, and spleen. (E) Cecum and colon MPO levels. *P < 0.05, significant difference from WT mice.
Figure 3
Figure 3. T5KO-H mice have increased permeability to macromolecules.
T5KO-L mice (noncolitic), T5KO-H mice (colitic), and WT littermates were fasted for 3 hours and then given FITC-dextran (4 kDa or 40 kDa) and HRP intragastrically as described in Methods. Three hours later, mice were bled, and serum concentrations of FITC and HRP were determined by fluorimetry and spectroscopy. *P < 0.05, significant difference from WT mice.
Figure 4
Figure 4. T5KO-L mice have an increased bacterial burden.
Feces or indicated tissue from T5KO-L (i.e., not robustly colitic) or WT littermates was homogenized and ambiently cultured on nonselective media. CFUs were quantitated as described in Methods. (A) Feces; (B) washed colons; (C) spleen; (D) liver. *P < 0.05, significant difference from WT mice.
Figure 5
Figure 5. T5KO-L mice show altered spontaneous colonic cytokine expression.
(A) Colons from WT or T5KO-L mice were cultured ex vivo for 24 hours in antibiotic-supplemented serum-free media. (A) Cytokines released into media measured by ELISA or SDS-PAGE immunoblotting (Ang4). ELISAs are shown as mean ± SEM of at least 8 mice per condition. (B) Sera from TLR5KO mice with low or high SAA (as defined in Figure 2) or WT littermates were assayed for cytokines as in A, but only KC and IL-18 were consistently detected. *P < 0.05, significant difference from WT mice.
Figure 6
Figure 6. TLR5/IL-10DKO mice show high colonic expression of IL-17 and severe colitis.
(A) TLR5/IL-10DKO mice and IL-10KO littermates were followed for incidence of rectal prolapse. Mice were euthanized upon developing prolapse, which always correlated with clear gross evidence of colitis. (B) Colons from indicated rectally prolapsed mice were isolated within 24 hours of prolapse and cultured in vitro for 24 hours; IL-17 production was measured by ELISA. *P < 0.05.
Figure 7
Figure 7. Deletion of TLR4 ablates colitis in TLR5KO mice.
Twelve-week-old TLR4/5DKO and WT littermates were euthanized and parameters assayed. H&E-stained (A) cecum and (B) colon. (C) Weights of the cecum, colon, and spleen. (D) Cecum and colonic MPO levels. (E) SAA levels. (F) Fecal and colon-adherent CFUs. *P < 0.05.
Figure 8
Figure 8. Deletion of TLR4 reduces elevated colonic cytokine production by TLR5KO mice.
(A and B) Colons from TLR4KO, TLR4/5DKO, or WT littermates were cultured ex vivo for 24 hours in antibiotic-supplemented serum-free media. (A) Cytokines released into media measured by ELISA. (B) Serum was assayed for KC. *P < 0.05, indicates significant difference from WT mice.
Figure 9
Figure 9. Antibiotic treatment ameliorates colitis in TLR5KO mice.
Five-week-old colitic T5KO-H mice were treated with streptomycin (1.25% in drinking water) for 3 weeks. (A) SAA levels before and after streptomycin treatment. (B) Representative image of gross appearance of cecum and colon. (i) Noncolitic (T5KO-L). (ii) Untreated colitic (T5KO-H). (iii) T5KO-H mice whose high SAA levels were lowered following 3 weeks of treatment with streptomycin.
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Comment in

  • J Clin Invest. 117:false.

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