Deletion of TLR5 results in spontaneous colitis in mice (original) (raw)
TLR5KO mice develop spontaneous colitis. Mice with a targeted deletion in TLR5 were generated on a mixed C57BL/6 × 129/Sv background at the Research Institute for Microbial Diseases, Suita, Osaka, Japan (10). Such mice appeared generally healthy, and study of the small intestine did not reveal gross phenotypic alterations. Mice that were heterozygous-null for TLR5 underwent quarantine at Charles River Laboratories, were transported to Emory University for further backcrossing to C57BL/6, and were then bred to make colonies of mice homozygous-null for TLR5 (TLR5KO) and WT littermates. That absence of both copies of the TLR5 gene resulted in loss of TLR5 function was verified by observing loss of serum cytokines in response to systemically administered flagellin. Specifically, as expected, TLR5KO mice exhibited dramatically reduced induction of serum cytokines such as keratinocyte-derived chemokine (KC) (murine homolog of IL-8) and IL-6 in response to systemically administered purified flagellin (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI33084DS1) in accordance with recent studies (10, 11). Interestingly, the level of KC induced by flagellin in C57BL/6 mice was higher than that induced by LPS, although flagellin, unlike LPS, failed to induce TNF-α (in WT or TLR5KO mice) (Supplemental Figure 2).
The majority of TLR5KO mice appeared to be generally healthy although they tended to be smaller than their age-matched, sex-matched WT littermates (body weights of 4-week-old females [g]: WT vs. KO, 16.3 ± 0.38 vs. 15.1 ± 0.24; males: WT vs. KO, 19.2 ± 0.41 vs. 17.6 ± 0.57; P < 0.05). However, the most striking phenotypic feature displayed by these mice was their frequency of rectal prolapse. Specifically, we observed that, at 3 levels of backcrossing (N4, N6, and N8), about 10%–12% mice lacking TLR5 but none of their WT littermates exhibited spontaneous rectal prolapse. Interestingly, prolapse has not been observed in TLR5KO mice maintained in Japan. In contrast with TLR5KO mice, mice lacking other TLRs (i.e., 2, 3, 4, 11) and the global TLR-signaling adapter MyD88 bred in our facility have not exhibited this phenotype whereas IL-10–null mice bred in our facility develop prolapse at the previously observed frequency (12). Rectal prolapse in TLR5KO mice was seemingly irreversible and correlated with unsuccessful breeding. Nonetheless, continued breeding of nonprolapsed TLR5KO mice produced offspring that continued to prolapse with substantial frequency (40 of 365) while offspring of their littermates did not show this phenotype (0 of >400) (Figure 1A).
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.
Rectal prolapse and reduced body weights are hallmarks of spontaneous murine colitis, and therefore we next determined whether TLR5KO mice were developing colitis as assessed by several well-defined indicators of this disease state. Specifically, excluding TLR5KO mice that had prolapsed (which all had robust transmural colitis), we compared TLR5KO mice with their WT littermates in terms of intestinal bleeding, morphology, organ weights, and histopathology. Additionally, we measured body weight and weights of their cecum, colon, liver, and spleen. Approximately 20% of the nonprolapsed TLR5KO mice exhibited detectable spontaneous bleeding (gross or occult), which was not detected in any WT mice. Moreover, 25% of TLR5KO mice displayed gross features of robust colitis including contracted ceca, swollen, opaque proximal colon, and absence of well-formed stools (Figure 1B). Such appearances were not exhibited by any of the WT mice. These alterations in the appearance of cecum and colon correlated with and thus could be quantitated by simply measuring organ weights, which were significantly altered in about 30% of the TLR5KO mice that had not exhibited spontaneous rectal prolapse when euthanized (Figure 1C). Further consistent with the colitic state, TLR5KO mice exhibited substantially enlarged spleens. Splenomegaly closely correlated with substantially enlarged mesenteric and linguinal lymph nodes (data not shown). Histopathologic analysis confirmed the notion that a portion of TLR5KO mice had colitis (regardless of whether they had rectal prolapse). Specifically, in mice displaying gross alterations in intestinal appearance (or organ weights), both the cecum and proximal colon displayed extensive areas of epithelial hyperplasia associated with mononuclear infiltrates, focal crypt epithelial destruction, and edema (Figure 1D), which was not present in WT littermates or in the 60% of TLR5KO mice whose intestines appeared normal by the above parameters. In accordance with histological appearance, use of a neutrophil-specific immunostain (α–GR-1) revealed large areas of neutrophils in the proximal colons of TLR5KO mice that were not observed in WT mice (Figure 1E) or in TLR5KO mice that lacked gross evidence of intestinal alterations. Together, these results indicate that approximately 35%–40% of TLR5KO mice were developing some degree of spontaneous colitis. In contrast to the cecum and colon, small intestines of TLR5KO mice appeared similar, both macroscopically and histopathologically, to those of WT littermates and did not exhibit a discernible difference in the number, size, or appearance of organized lymphoid structures in these tissues (Supplemental Figure 3).
To facilitate our understanding of how loss of TLR5 might result in spontaneous colitis, we sought to define a serum marker that would allow us to select mice that had not yet developed robust colitis. We observed that levels of serum amyloid A (SAA), a general inflammatory acute phase marker that has been shown to correlate with degree of intestinal inflammation in humans and mice (13, 14), could be used to identify mice with robust colitis. Specifically, whereas all WT mice assayed had SAA levels lower than 40 μg/ml, 41% (29/70) of TLR5KO mice displayed SAA levels of over 50 μg/ml (Figure 2A). Mice with elevated SAA also displayed elevated levels of serum lipocalin 2 (Figure 2B), which is another general marker of inflammation whose serum levels are elevated in colitis (15). SAA was most elevated in mice that had developed rectal prolapse, with every prolapsed mouse having an SAA concentration higher than 2000 μg/ml. Further, TLR5KO mice with SAA levels greater than 100 μg/ml displayed gross clear evidence of colitis by the above-defined parameters whereas there were only modest differences in these parameters between WT mice and TLR5KO mice with low SAA levels (T5KO-L) (Figure 2, C–E). SAA levels also correlated with the degree of colitis determined by histopathologic assessment. Specifically, whereas histopathologic scoring showed only modest differences between WT and T5KO-L mice, TLR5KO mice with high SAA levels (T5KO-H) had colitis somewhat similar to that exhibited by IL-10KO mice, particularly IL-10KO mice that were treated with piroxicam, which promotes their development of colitis (16; Table 1). Further consistent with the colitic state, T5KO-H mice also had a significant degree of anemia and leukocytosis (Table 2), while T5KO-L mice did not differ significantly from WT littermates. Thus, SAA levels can be used to stratify TLR5KO mice as likely having or not having robust colitis. When measured shortly after weaning (25 days), 0 of 28 TLR5KO mice had elevated SAA. Rather, SAA became elevated in some mice between 5 and 8 weeks of age while some mice maintained low SAA even at 6 months. Thus, while some T5KO-L mice may ultimately develop colitis, the designation also reflects a degree of disease heterogeneity exhibited by these mice.
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.
Histopathologic scoring of colitis
T5KO-H mice exhibit anemia and leukocytosis
TLR5KO mice have an increased burden of commensal bacteria. We next considered some potential mechanisms that might underlie the colitis exhibited by TLR5KO mice. Intestinal barrier dysfunction is associated with and suggested to underlie some cases of inflammatory bowel disease in humans and the spontaneous colitis exhibited by Mdr-null mice (17). Thus, we measured epithelial barrier function by orally gavaging mice with commonly used permeability markers (HRP and FITC-dextran) and measuring their levels in the serum. We observed that robustly colitic TLR5KO mice (i.e., T5KO-H) exhibited increased intestinal permeability to these macromolecules (Figure 3). This approach did not reveal a difference in permeability between T5KO-L mice and WT littermates. Thus, increased gut permeability to macromolecules occurs in TLR5KO colitis but may not be an early event in the disease process.
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.
Henceforth, we focused our studies on noncolitic TLR5KO mice in the hope of observing events that might drive their tendency to develop colitis. We considered whether loss of TLR5 might make mice incapable of maintaining proper homeostasis of their intestinal microflora. To begin to address this possibility, we quantitated the levels of gut bacteria that were able to grow on nonselective agar in ambient conditions. We observed that T5KO-L mice exhibited moderately elevated (about 5-fold) numbers of total culturable bacteria in their feces (Figure 4A). The degree of elevation was more pronounced in T5KO-H mice although this group also exhibited greater variability (by geometric mean, level of total culturable fecal CFUs in T5KO-H mice was 8.5 ± 5.5–fold higher than in T5KO-L mice). Next, we quantitated levels of bacteria in colonic segments from which we had first removed nonadherent material (as described in Methods). We observed markedly higher levels of such tightly adherent bacteria in colons of T5KO-L mice (Figure 4B). Furthermore, a number of T5KO-L mice but not their WT littermates had a modest but nonetheless clearly detectable bacterial burden in both their livers and spleens of approximately 100–200 bacteria per organ (Figure 4, C and D). Because the vast majority of intestinal bacteria are strict anaerobes and quite difficult, if not impossible, to culture, these results should be viewed as only a small sampling of the gut microflora. Nonetheless, they suggest loss of TLR5 results in significant alterations in the gut microflora.
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.
We next sought to determine whether the bacteria we cultured from the liver/spleen of T5KO-L mice might be known pathogens or whether they were simply normal gut bacteria that may have translocated from the gut. We noted that the colonies of bacteria cultured from these organs appeared either small and whitish-yellow or large and golden. Colonies of similar appearance could be found in the fecal cultures of both WT and T5KO-L mice although the vast majority of fecal CFUs produce much smaller, paler colonies. Thus, we hypothesized that the fecal CFUs that formed such colonies may be the same species as the CFUs we cultured from the liver and spleen. To investigate this possibility, we selected colonies of each type from the feces or organs and examined their appearance by gram staining. All were gram-positive cocci, and colony appearance correlated with their clustering style. Next, we cloned and sequenced the 16S RNAs from a small number of these isolates. Of the 6 T5KO-L liver/spleen isolates that were sequenced, 4 were Staphylococcus saprophyticus and 2 were Staphylococcus hominis. Of the 2 WT fecal CFU isolates that were sequenced, 1 was S. saprophyticus and 1 was S. hominis. Identifying these strains in the feces of WT mice is consistent with the knowledge that these bacteria are normal components of the intestinal microflora of cattle, rodents, and humans (18). While identifying these few CFUs as nonpathogenic does not, of course, rule out the possible presence of a pathogen in our colony, that immunodeficient mice (RAG-1KO and MyD88KO) that have shared cages and bred with these TLR5KO mice have not developed any clinical indicators of disease argues against a typical pathogen. Furthermore, the failure of the WT littermates of T5KO to exhibit indications of colitis at 3 levels of interbreeding/backcrossing suggests that the gut bacteria in these mice are most likely commensal microbes and argues against the presence of a truly pathogenic microbe in our colony. Thus, while Staphylococci attaining an extraintestinal localization is likely a consequence of the loss of TLR5 signaling, neither these isolates nor other bacteria colonizing these mice are pathogenic in WT mice.
TLR5KO colons exhibit dysregulated gene expression. We reasoned that reduced expression of host defense genes due to the loss of TLR5 signaling might underlie the increased bacterial burden of T5KO-L mice, although, conversely, it also seemed quite possible that the increased bacterial burden might result in greater expression of host defense genes due to increased activation of other innate immune signaling pathways. To investigate these possibilities, we used a recently described protocol (1) to assess the ongoing intestinal production of cytokines by 24-hour ex vivo culture of colonic intestinal segments. We measured cytokines that are potently induced by flagellin (KC, IL-6) as well as cytokines that are not substantially induced (at least in our hands) by purified flagellin (IFN-γ, IL-12p70, IL-23, IL-1β, and TNF-α) but which are potently upregulated in response to the TLR4 ligand LPS. We observed that, despite their increased bacterial burden, T5KO-L mice made markedly lower levels of the flagellin-induced cytokines KC and IL-6 (Figure 5A). In contrast, despite lack of histopathological evidence of inflammation, T5KO-L mice colons released markedly higher levels of proinflammatory cytokines (IL-1β and TNF-α), Th1 cytokines (IFN-γ and IL-12p70), and Th17 cytokines (IL-17 and IL-23). We also assayed these colonic supernatants for levels of the protein angiogenin-4 (Ang4), which is thought to play an important role in host defense against gram-positive bacteria (19). Ang4 expression was markedly reduced in the colons of T5KO-L mice compared with WT littermates. Last, we also measured the levels of these cytokines in the serum of these mice. While most of these cytokines were undetectable in serum of T5KO-L mice (i.e., not robustly colitic), these animals had lower serum KC than WT littermates while colitic T5KO-H mice had higher serum levels of this chemokine compared with WT littermates (Figure 5B). Serum IL-18 was elevated in some TLR5KO mice. Thus, loss of TLR5 resulted in reduced expression of some host defense genes, particularly Ang4 and 2 cytokines (KC and IL-6) that are robustly induced by flagellin. However, loss of TLR5 also resulted in increased expression of a panel of proinflammatory cytokines whose expression may be driven by other TLR ligands. To further assess altered gene expression in the colons in TLR5KO mice, we next performed a global assay of gene expression. Specifically, we used cDNA microarray to compare the relative gene expression in the colons of TLR5KO mice (T5KO-L or T5KO-H) with that of their WT littermates. These studies pooled mRNA isolated from 3 mice per group. Such analysis may reflect changes in gene expression in resident cells in the intestine and/or reflect infiltration of leukocytes, particularly in the case of the overtly colitic mice. Compared with their WT littermates, robustly colitic T5KO-H mice exhibited a significant elevation of more than 800 (of 35,000) genes assayed. While the overall profile of these genes might most simply be described as “proinflammatory,” they can also be reasonably classified into functional categories (Table 3; specific genes are listed in Supplemental Table 1) including antibacterial genes and cytokines/chemokines. Qualitatively, a similar pattern of colonic gene expression was observed in T5KO-L mice although there was a reduction both in the number of genes in each category that were significantly elevated and the extent to which their expression was elevated. The increased expression of a number of these mRNAs agreed with our protein assays of colon cultures (performed on at least 5 mice per group). Subsequently, such samples were assayed for several other proteins (IL-10, IL-18, MUC2, MMP7, MMP9), which were all upregulated in the colons of TLR5KO mice in accordance with our microarray analysis (Supplemental Figure 4). This microarray analysis also indicated that TLR5KO mice had increased expression of genes involved in LPS signaling, for example, TLR4, LPS-binding protein, and CD14 (Supplemental Table 1). The upregulation of TLR4 was confirmed by quantitative RT-PCR (Supplemental Figure 4).
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.
Summary of cDNA microarray analysis
IL-10 functions to reduce incidence/severity of TLR5KO colitis. The above-described studies of intestinal gene expression indicate that even T5KO mice that lack histopathological evidence of colitis have dysregulated intestinal gene expression. We hypothesized that endogenous antiinflammatory mechanisms may function to prevent spontaneous colitis. For example, expression of the antiinflammatory cytokine IL-10 is elevated 2-fold in noncolitic T5KO-L and elevated 8-fold in colitic T5KO-H mice (Supplemental Figure 4 and Supplemental Table 1), an expression pattern that suggests it may be functioning to counterregulate the elevated proinflammatory gene expression exhibited by these mice. To investigate this possibility, TLR5KO mice were crossed with mice lacking the gene for IL-10 to generate TLR5/IL-10DKO (DKO, double KO) mice and IL-10KO littermates. IL-10–deficient littermates exhibited rectal prolapse at a rate similar to our original colony of IL-10KO (about 50% by 16 weeks), with the remainder being sufficiently healthy to produce offspring. In contrast, deletion of TLR5 on an IL-10–null background resulted in 80% rectal prolapse by 10 weeks (100% by 6 months) and a uniform failure to breed (Figure 6A). Furthermore, T5/IL-10DKO mice showed a striking elevation of colonic IL-17 production whereas colitic IL-10KO mice expressed much less IL-17 than colitic T5KO mice (Figure 6B). These results suggest that the IL-10 antiinflammatory pathway plays an important role in counterregulating the proinflammatory pathways that result from loss of TLR5 but can only prevent colitis in a portion of these mice. Furthermore, the high amount of IL-17 made by colitic TLR5KO relative to colitic IL-10KO mice suggests a potential role for the Th17 pathway in TLR5KO colitis.
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.
TLR5KO colitis is driven by activation of TLR4. TLR5 signaling is thought to be entirely MyD88 dependent, and thus, MyD88KO mice are completely deficient in TLR5 function. However, MyD88KO mice do not develop spontaneous colitis (even when sharing cages with TLR5KO mice in our facility). This suggests that the increased bacterial burden of TLR5KO mice may result in colitis via activation of other TLRs — a notion also suggested by our microarray data. In support of this possibility, colon cultures of MyD88KO mice lacked all cytokines measured, including KC, TNF-α, and IL-1β (data not shown). Thus, we generated mice lacking both TLR4 and TLR5 and examined their intestinal phenotype. In contrast to TLR5KO mice, TLR4/5DKO mice neither developed rectal prolapse (0 of 75 — of which many were followed up to 10 months of age) nor showed detectable intestinal bleeding. Further, microscopic examination of the cecum and colon revealed only slight histopathologic abnormalities in the cecum (Table 1 and Figure 7, A and B). TLR4/5DKO mice had only a slight increase in colon weight and lacked the reduction in cecum weight and splenomegaly (and enlarged intestinal lymph nodes) that was prominent in TLR5KO mice (Figure 7C). Myeloperoxidase (MPO) levels were moderately elevated in the cecum but not in the colon (Figure 7B), and some TLR4/5DKO mice (7 of 25) had significantly elevated levels of SAA (Figure 7C). Such lack of evidence of colitis was observed in TLR4/5DKO mice at 12 weeks and 6 months of age. We also determined whether TLR4/5DKO mice had the elevated bacterial burdens that are exhibited by TLR5KO mice. We observed that, like TLR5KO mice, TLR4/5DKO mice had a significantly elevated level of culturable gut bacteria both in their feces and adherent to their colons (Figure 7D). However, unlike TLR5KO mice, TLR4/5DKO mice lacked detectable bacteria in the liver/spleen, suggesting that translocation of intestinal bacteria to these organs may occur consequent to colonic inflammation. We next compared spontaneous cytokine secretion from the colons of these mice (Figure 8). In order to both allow direct comparison with littermates and evaluate results relative to the above data, in Figure 8, we maintained the same y axis scale as utilized in Figure 7. Despite an elevated intestinal bacterial load, colons from TLR4/5DKO mice exhibited less spontaneous secretion of KC, IL-6, and TNF-α and, like WT mice, did not secrete detectable levels of IL-12p70 or IL-23. Furthermore, TLR4/5DKO mice had markedly lower levels of serum KC. To further evaluate the role of TLR5 in gut cytokine production, we also measured colonic cytokine production in mice lacking TLR4 (offspring of littermates of TLR4/5DKO). We observed that loss of TLR5 on a TLR4-null background still lowered colonic production of KC and IL-6 but resulted in only a modest increase in TNF-α and IL-17. TLR4/5DKO mice had a level of IL-1β (Figure 8) secretion that was still elevated relative to WT littermates, suggesting this may be driven by another pathway. Last, although consistently reduced in TLR5KO mice, Ang4 expression was variable in TLR4/5DKO mice (data not shown), suggesting the possibility that this gene’s expression may be regulated by multiple pathways and/or that its reduction in TLR5KO may in fact result from the cytokine/inflammatory environment.
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.
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.
As an additional approach, we sought to lower levels of TLR ligands via reducing levels of enteric bacteria by treating mice with antibiotics. Specifically, we next treated 5-week-old colitic TLR5KO mice for 3 weeks with streptomycin and determined whether it ameliorated their colitis. Such treatment dramatically lowered their SAA levels (Figure 9) and reduced robust inflammation and rectal prolapse (prevented prolapse in 5 of 7 treated mice). In contrast, untreated colitic TLR5KO mice uniformly (18 of 18) developed rectal prolapse within this time frame when not treated with antibiotics. Furthermore, we similarly treated TLR5KO mice that had already developed rectal prolapse and found that such treated mice had intestines and spleens of relatively normal size and appearance although such treatment did not reverse prolapse (data not shown). Together, these results suggest that loss of TLR5 results in inability to manage gut microflora, resulting in increased activation of TLR4 and likely other pathways that mediate the proinflammatory gene expression that drives recruitment of inflammatory cells, which allows intestinal bacteria to reach the spleen.
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.
To further determine whether loss of TLR5 results in elevated responses through TLR4, we measured LPS-induced responses ex vivo of splenocytes and peritoneal macrophages. As compared with WT littermates, splenocytes from noncolitic (i.e., T5KO-L) TLR5KO mice exhibited a 3-fold higher level of IFN-γ production in response to LPS (Supplemental Figure 5A). In contrast, macrophages isolated from WT and TLR5KO mice responded similarly to LPS (Supplemental Figure 5B), indicating that elevated response of TLR5KO splenocytes to LPS is likely acquired and may manifest in the intestine due to the altered gut bacterial homeostasis rather than reflect an inherent role of TLR5 in regulating TLR4.