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Cited by 214Inflammatory Bowel DiseaseGut microbiota and healthImmune Response and Inflammation

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Shift Towards Pro-inflammatory Intestinal Bacteria Aggravates Acute Murine Colitis via Toll-like Receptors 2 and 4

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Heimesaat, Markus M., Fischer, André, Siegmund, Britta, Kupz, Andreas, Niebergall, Julia, Fuchs, David, Jahn, Hannah-Katharina, Freudenberg, Marina, Loddenkemper, Christoph, Batra, Arvind, Lehr, Hans-Anton, Liesenfeld, Oliver, Blaut, Michael, Göbel, Ulf B., Schumann, Ralf R., Bereswill, Stefan·2007·PLoS ONE
DOI 10.1371/journal.pone.0000662PMC PMC1914380
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Abstract

BACKGROUND: Gut bacteria trigger colitis in animal models and are suspected to aggravate inflammatory bowel diseases. We have recently reported that Escherichia coli accumulates in murine ileitis and exacerbates small intestinal inflammation via Toll-like receptor (TLR) signaling. METHODOLOGY AND PRINCIPAL FINDINGS: Because knowledge on shifts in the intestinal microflora during colitis is limited, we performed a global survey of the colon flora of C57BL/10 wild-type (wt), TLR2(-/-), TLR4(-/-), and TLR2/4(-/-) mice treated for seven days with 3.5% dextrane-sulfate-sodium (DSS). As compared to wt animals, TLR2(-/-), TLR4(-/-), and TLR2/4(-/-) mice displayed reduced macroscopic signs of acute colitis and the amelioration of inflammation was associated with reduced IFN-gamma levels in mesenteric lymph nodes, lower amounts of neutrophils, and less FOXP3-positive T-cells in the colon in situ. During acute colitis E. coli increased in wt and TLR-deficient mice (P<0.05), but the final numbers reached were significantly lower in TLR2(-/-), TLR4(-/-) and TLR2/4(-/-) animals, as compared to wt controls (P<0.01). Concentrations of Bacteroides/ Prevotella spp., and enterococci did not increase during colitis, but their numbers were significantly reduced in the colon of DSS-treated TLR2/4(-/-) animals (P<0.01). Numbers of lactobacilli and clostridia remained unaffected by colitis, irrespective of the TLR-genotype of mice. Culture-independent molecular analyses confirmed the microflora shifts towards enterobacteria during colitis and showed that the gut flora composition was similar in both, healthy wt and TLR-deficient animals. CONCLUSIONS AND SIGNIFICANCE: DSS-induced colitis is characterized by a shift in the intestinal microflora towards pro-inflammatory Gram-negative bacteria. Bacterial products exacerbate acute inflammation via TLR2- and TLR4-signaling and direct the recruitment of neutrophils and regulatory T-cells to intestinal sites. E. coli may serve as a biomarker for colitis severity and DSS-induced barrier damage seems to be a valuable model to further identify bacterial factors involved in maintaining intestinal homeostasis and to test therapeutic interventions based upon anti-TLR strategies.

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Figure 1 Colitis severity and inflammatory responses in TLR2-/-, TLR4-/-, and TLR2/4-/- mice, as compared to wt controls. (A) Severity of colitis in DSS-treated animals. Clinical outcome of colitis was recorded in wt mice (WT, n = 10) and in mice lacking TLR2 (TLR2-/-, n = 10), TLR4 (TLR4-/-, n = 12), or both (TLR2/4-/-, n = 11). Clinical parameters of colitis were monitored daily (see methods). The data sets from two independent experiments were pooled. Total colitis scores were analyzed at day eight after DSS-treatment for seven days. (B) IFN-gamma concentrations in supernatants of colonic MLN cultures from wt and TLR-deficient animals. Supernatants from MLN cultures of wt mice (WT, black bars, n = 3), and animals lacking TLR2 (TLR2-/-, n = 4), TLR4 (TLR4-/-, n = 5), or both (TLR2/4-/-, n = 4) with colitis were analyzed at day eight after DSS-treatment for seven days. (A/B) Mean values, standard deviations and significance levels (as compared to the wt animals) determined by Student's t-test are indicated (***, P<0.001).
Figure 1 Colitis severity and inflammatory responses in TLR2-/-, TLR4-/-, and TLR2/4-/- mice, as compared to wt controls. (A) Severity of colitis in DSS-treated animals. Clinical outcome of colitis was recorded in wt mice (WT, n = 10) and in mice lacking TLR2 (TLR2-/-, n = 10), TLR4 (TLR4-/-, n = 12), or both (TLR2/4-/-, n = 11). Clinical parameters of colitis were monitored daily (see methods). The data sets from two independent experiments were pooled. Total colitis scores were analyzed at day eight after DSS-treatment for seven days. (B) IFN-gamma concentrations in supernatants of colonic MLN cultures from wt and TLR-deficient animals. Supernatants from MLN cultures of wt mice (WT, black bars, n = 3), and animals lacking TLR2 (TLR2-/-, n = 4), TLR4 (TLR4-/-, n = 5), or both (TLR2/4-/-, n = 4) with colitis were analyzed at day eight after DSS-treatment for seven days. (A/B) Mean values, standard deviations and significance levels (as compared to the wt animals) determined by Student's t-test are indicated (***, P<0.001).CC BY · PubMed Central
Figure 2 Quantification of immune cells in the colon of DSS-treated mice. Numbers of T-cells, regulatory T-cells, and neutrophils in the colon of DSS-treated wt mice and mice lacking TLR2 and/ or TLR4. The average number of cells positive for CD3, FOXP3, or myeloperoxidase from at least five high power fields (HPF, 400×magnification) per animal were determined microscopically in immunostained colon sections of wt mice (WT), and animals lacking TLR2 (TLR2-/-), TLR4-/- (TLR4-/-), or both (TLR2/4-/-) at day eight after DSS-treatment for seven days (C, grey circles). Healthy animals served as controls (H, white circles). The numbers of analyzed animals are given in parenthesis. Mean values (black bars), and significance levels (as compared to wt animals with colitis) determined by Student's t-test are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).
Figure 2 Quantification of immune cells in the colon of DSS-treated mice. Numbers of T-cells, regulatory T-cells, and neutrophils in the colon of DSS-treated wt mice and mice lacking TLR2 and/ or TLR4. The average number of cells positive for CD3, FOXP3, or myeloperoxidase from at least five high power fields (HPF, 400×magnification) per animal were determined microscopically in immunostained colon sections of wt mice (WT), and animals lacking TLR2 (TLR2-/-), TLR4-/- (TLR4-/-), or both (TLR2/4-/-) at day eight after DSS-treatment for seven days (C, grey circles). Healthy animals served as controls (H, white circles). The numbers of analyzed animals are given in parenthesis. Mean values (black bars), and significance levels (as compared to wt animals with colitis) determined by Student's t-test are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).CC BY · PubMed Central
Figure 3 Molecular gut flora analysis in mice lacking TLR2 and/or TLR4. Detailed molecular investigations of the colon microbiota in three months old C57BL/10 mice lacking TLR2, TLR4, or both were performed by analysis of 16S rRNA gene libraries (A) or high-resolution DGGE (B) with primers HDA-1-GC and HDA-2 (see methods). (A) Results from gene library analysis. Complete 16S rRNA genes were amplified from total luminal colon content DNA isolated from wt mice (WT, n = 3), and from mice (n = 3 each group) lacking TLR2 (TLR2-/-), TLR4 (TLR4-/-), or both (TLR2/4-/-). The amplicons were cloned and sequenced as described in the Methods section. Bars represent percentage amounts of gut bacterial groups indicated on the Y-axis. (B) Results from DGGE analysis of PCR-amplified total bacterial 16S rRNA gene fragments. Each lane shows DGGE profiles of the bacterial flora from the colon of healthy wt mice, and animals lacking TLR2 (TLR2-/-), TLR4 (TLR4-/-), or both (TLR2/4-/-). Mice of identical age (three months) originated from two different animal facilities (as indicated above the lanes). DNA bands, which were absent or weak in DGGE profiles from TLR4-/- and TLR2/4-/- animals are marked by grey arrows. Bacterial species identified by sequence analysis of 16S rRNA gene fragments in the corresponding bands are indicated on the left. Total numbers of DGGE bands in the profile from wt animals were counted and set to 100% in the reference pattern (RP). Presence and absence of bands in profiles from the respective TLR-deficient animals was recorded and similarity values were calculated and expressed as % similarity to the wt RP. The DGGE profiles are representative for at least three mice per group and experiment. Results were reproduced in two independent experiments.
Figure 3 Molecular gut flora analysis in mice lacking TLR2 and/or TLR4. Detailed molecular investigations of the colon microbiota in three months old C57BL/10 mice lacking TLR2, TLR4, or both were performed by analysis of 16S rRNA gene libraries (A) or high-resolution DGGE (B) with primers HDA-1-GC and HDA-2 (see methods). (A) Results from gene library analysis. Complete 16S rRNA genes were amplified from total luminal colon content DNA isolated from wt mice (WT, n = 3), and from mice (n = 3 each group) lacking TLR2 (TLR2-/-), TLR4 (TLR4-/-), or both (TLR2/4-/-). The amplicons were cloned and sequenced as described in the Methods section. Bars represent percentage amounts of gut bacterial groups indicated on the Y-axis. (B) Results from DGGE analysis of PCR-amplified total bacterial 16S rRNA gene fragments. Each lane shows DGGE profiles of the bacterial flora from the colon of healthy wt mice, and animals lacking TLR2 (TLR2-/-), TLR4 (TLR4-/-), or both (TLR2/4-/-). Mice of identical age (three months) originated from two different animal facilities (as indicated above the lanes). DNA bands, which were absent or weak in DGGE profiles from TLR4-/- and TLR2/4-/- animals are marked by grey arrows. Bacterial species identified by sequence analysis of 16S rRNA gene fragments in the corresponding bands are indicated on the left. Total numbers of DGGE bands in the profile from wt animals were counted and set to 100% in the reference pattern (RP). Presence and absence of bands in profiles from the respective TLR-deficient animals was recorded and similarity values were calculated and expressed as % similarity to the wt RP. The DGGE profiles are representative for at least three mice per group and experiment. Results were reproduced in two independent experiments.CC BY · PubMed Central
Figure 4 Molecular analysis of predominant bacterial communities in the colon of healthy and diseased mice. (A) DGGE profiles of the colon flora in healthy and diseased wt mice. Results are shown for C57BL/10 wt mice without DSS-treatment or at day eight after DSS-treatment for seven days (as indicated above the lanes). Bacterial species were identified by comparative sequence analyses of 16S rRNA gene fragments amplified from DNA eluted from respective DNA bands. The DGGE profiles are representative for at least three mice per group and experiment. (B) DGGE profiles of the colon flora in healthy and diseased TLR2/4-/- mice. DGGE profiles of luminal intestinal bacteria from wt and TLR2/4-/- mice were generated by PCR-based analysis of total DNA from colon contents of mice with or without colitis at day eight after DSS-treatment for seven days (as indicated). Bacterial species were identified by comparative sequence analysis of 16S rRNA gene fragments amplified from DNA eluted from respective DNA bands. The DGGE profiles are representative for at least three mice per group and experiment. (A/B) Primers GC968F and R1378 (Heimesaat et al., 2006) were used for amplification. Results were reproduced in at least two independent experiments.
Figure 4 Molecular analysis of predominant bacterial communities in the colon of healthy and diseased mice. (A) DGGE profiles of the colon flora in healthy and diseased wt mice. Results are shown for C57BL/10 wt mice without DSS-treatment or at day eight after DSS-treatment for seven days (as indicated above the lanes). Bacterial species were identified by comparative sequence analyses of 16S rRNA gene fragments amplified from DNA eluted from respective DNA bands. The DGGE profiles are representative for at least three mice per group and experiment. (B) DGGE profiles of the colon flora in healthy and diseased TLR2/4-/- mice. DGGE profiles of luminal intestinal bacteria from wt and TLR2/4-/- mice were generated by PCR-based analysis of total DNA from colon contents of mice with or without colitis at day eight after DSS-treatment for seven days (as indicated). Bacterial species were identified by comparative sequence analysis of 16S rRNA gene fragments amplified from DNA eluted from respective DNA bands. The DGGE profiles are representative for at least three mice per group and experiment. (A/B) Primers GC968F and R1378 (Heimesaat et al., 2006) were used for amplification. Results were reproduced in at least two independent experiments.CC BY · PubMed Central
Figure 5 Cultural analysis of gut flora changes in healthy and diseased TLR2-/-, TLR4-/-, and TLR2/4-/- mice, as compared to wt controls. Bacterial counts of (A) Gram-negative (E. coli, Bacteroides/ Prevotella spp.) and (B) Gram-positive bacteria (Enterococcus spp., clostridia and lactobacilli) in luminal colon contents from healthy animals (H) and from mice with colitis at day eight after DSS-treatment for seven days (C) as determined by culture. Bacterial species were identified by biochemical analysis and reconfirmed by comparative sequence analyses of 16S rRNA genes. Results are shown for wt mice (WT, above) and for mice lacking TLR2 (TLR2-/-), TLR4 (TLR4-/-), or both (TLR2/4-/-) as indicated on the x-axis. Animal numbers analyzed are given in parentheses. The data sets were pooled from two independent experiments. The boxes represent 75th % and 25th % percentiles of medians (indicated by black bars within the boxes. Maximum and minimum values are indicated by lines above and below boxes, respectively. Significance levels were determined by Mann-Whitney-U-Test. Underlined P values shown in black were calculated from comparisons of healthy versus diseased animals. P values shown in grey were calculated from the bacterial number in diseased animals (all versus WT).
Figure 5 Cultural analysis of gut flora changes in healthy and diseased TLR2-/-, TLR4-/-, and TLR2/4-/- mice, as compared to wt controls. Bacterial counts of (A) Gram-negative (E. coli, Bacteroides/ Prevotella spp.) and (B) Gram-positive bacteria (Enterococcus spp., clostridia and lactobacilli) in luminal colon contents from healthy animals (H) and from mice with colitis at day eight after DSS-treatment for seven days (C) as determined by culture. Bacterial species were identified by biochemical analysis and reconfirmed by comparative sequence analyses of 16S rRNA genes. Results are shown for wt mice (WT, above) and for mice lacking TLR2 (TLR2-/-), TLR4 (TLR4-/-), or both (TLR2/4-/-) as indicated on the x-axis. Animal numbers analyzed are given in parentheses. The data sets were pooled from two independent experiments. The boxes represent 75th % and 25th % percentiles of medians (indicated by black bars within the boxes. Maximum and minimum values are indicated by lines above and below boxes, respectively. Significance levels were determined by Mann-Whitney-U-Test. Underlined P values shown in black were calculated from comparisons of healthy versus diseased animals. P values shown in grey were calculated from the bacterial number in diseased animals (all versus WT).CC BY · PubMed Central

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Authors
Heimesaat, M. M., Fischer, A., Siegmund, B., Kupz, A., Niebergall, J., Fuchs, D., Jahn, H.-K., Freudenberg, M., Loddenkemper, C., Batra, A., Lehr, H.-A., Liesenfeld, O., Blaut, M., Göbel, U. B., Schumann, R. R., & Bereswill, S.
Affiliations
  • Charité – Universitätsmedizin Berlin (DE)
  • Max Planck Society (DE)
  • Centre Hospitalier Universitaire Vaudois (CH)
Corresponding author
Bereswill, Stefan
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2007-07-25
Volume
2
Issue
7
Pages
e662
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0000662
PMC
PMC1914380
OpenAlex
W2065205410
Cited by
213
References
36
Citations by year
2026: 3 · 2025: 2 · 2024: 4 · 2023: 10 · 2022: 6 · 2021: 11 · 2020: 7 · 2019: 12 · 2018: 7 · 2017: 11 · 2016: 8 · 2015: 16 · 2014: 16 · 2013: 16 · 2012: 26
FWCI
6.09
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • SERVAL (Université de Lausanne) (publishedVersion, other-oa)
  • ResearchOnline - JCU (James Cook University) (submittedVersion)
  • IRIS (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Max Planck Digital Library (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Funders
  • Sonnenfeld Stiftung
  • Deutsche Forschungsgemeinschaft (KFO104, SFB633, Project 6, SPP1110)
Topics
  • Health Sciences › Medicine › Gastroenterology › Inflammatory Bowel Disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
acute colitis, gut microbiota, TLR2, TLR4, Escherichia coli, DSS-induced colitis, Gram-negative bacteria, neutrophil recruitment, regulatory T cells, interferon-gamma, Bacteroides-Prevotella, Enterococcus, intestinal barrier injury, inflammatory bowel disease
MeSH terms
Acute Disease, Animals, Bacterial Infections, Colitis, Colon, Humans, Inflammation, Mice, Inbred C57BL, Neutrophils, Inflammatory Bowel Diseases, Signal Transduction, Toll-Like Receptor 2, Toll-Like Receptor 4, Mice

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