A Novel Mouse Model of <i>Campylobacter jejuni</i> Gastroenteritis Reveals Key Pro-inflammatory and Tissue Protective Roles for Toll-like Receptor Signaling during Infection

<div><p><i>Campylobacter jejuni</i> is a major source of foodborne illness in the developed world, and a common cause of clinical gastroenteritis. Exactly how <i>C. jejuni</i> colonizes its host's intestines and causes disease is poorly understood. Although it causes severe diarrhea and gastroenteritis in humans, <i>C. jejuni</i> typically dwells as a commensal microbe within the intestines of most animals, including birds, where its colonization is asymptomatic. Pretreatment of C57BL/6 mice with the antibiotic vancomycin facilitated intestinal <i>C. jejuni</i> colonization, albeit with minimal pathology. In contrast, vancomycin pretreatment of mice deficient in SIGIRR (<i>Sigirr^−/−</i>), a negative regulator of MyD88-dependent signaling led to heavy and widespread <i>C. jejuni</i> colonization, accompanied by severe gastroenteritis involving strongly elevated transcription of Th1/Th17 cytokines. <i>C. jejuni</i> heavily colonized the cecal and colonic crypts of <i>Sigirr^−/−</i> mice, adhering to, as well as invading intestinal epithelial cells. This infectivity was dependent on established <i>C. jejuni</i> pathogenicity factors, capsular polysaccharides (<i>kpsM</i>) and motility/flagella (<i>flaA</i>). We also explored the basis for the inflammatory response elicited by <i>C. jejuni</i> in <i>Sigirr^−/−</i> mice, focusing on the roles played by Toll-like receptors (TLR) 2 and 4, as these innate receptors were strongly stimulated by <i>C. jejuni</i>. Despite heavy colonization, <i>Tlr4^−/−/Sigirr^−/−</i> mice were largely unresponsive to infection by <i>C. jejuni</i>, whereas <i>Tlr2^−/−/Sigirr^−/−</i> mice developed exaggerated inflammation and pathology. This indicates that TLR4 signaling underlies the majority of the enteritis seen in this model, whereas TLR2 signaling had a protective role, acting to promote mucosal integrity. Furthermore, we found that loss of the <i>C. jejuni</i> capsule led to increased TLR4 activation and exaggerated inflammation and gastroenteritis. Together, these results validate the use of <i>Sigirr^−/−</i> mice as an exciting and relevant animal model for studying the pathogenesis and innate immune responses to <i>C. jejuni</i>.</p></div

TLR2 and 4 reporter assays.

<p>HEK-Blue hTLR2 (A) and HEK-Blue hTLR4 (B) reporter cell lines were exposed for 4 hrs to either live and heat-killed wildtype <i>C. jejuni</i> 81–176, <i>ΔkpsM</i> or <i>ΔkpsM+kpsM</i>. The wild-type <i>C. jejuni</i> stimulates both TLR2 and TLR4 in a dose-dependent fashion. The <i>ΔkpsM</i> mutant significantly increased the signaling by both TLR2 and TLR4, as indicated by the assay, with the increase in stimulation also being in a dose-dependent manner, except for the TLR4 assay where the readers were near the maximum for both the 20 and 200 MOI readings. The complemented <i>ΔkpsM+kpsM</i> strain completely restored the wild-type phenotype with TLR2 and mostly restored the phenotype with TLR4. Values represent the mean of three independent experiments and statistical significance was determined by a two-way ANOVA with a Bonferroni post-test. (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).</p

Colonization of WT and <i>Sigirr−/−</i> mice by <i>C. jejuni</i> 81–176, 3 and 7 DPI.

<p>(A) High numbers (∼10⁹ CFUs/g) of <i>C. jejuni</i> were recovered at both 3 and 7 DPI from the ceca of infected mice that were pre-treated with 5 mg of vancomycin. No statistically significant differences in numbers were found between WT and <i>Sigirr^−/−</i> mice as indicated by a t-test, p>0.05. n = 10 or 11 WT mice, and 12 or 13 <i>Sigirr^−/−</i> mice for 3 and 7 DPI respectively. (B) H&E stained, formalin-fixed histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice 3 and 7 DPI. Upper panels are ×100 magnification, while lower panels are ×400 magnification. (C) Pathological scoring was done by two blinded observers, using H&E stained, formalin-fixed cecal tissue sections. Each condition represents a minimum of three separate experimental replicates, with 2–3 mice per experiment for a total of 6–9 mice per group. Control mice were used as a reference and consisted of 3 uninfected mice, pre-treated with a single dose of 5 mg/100 µl vancomycin, and euthanized 3 days post-treatment. WT (B6) mice did not exhibit any significant signs of inflammation, while <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice showed a significant increase relative to the uninfected <i>Sigirr^−/−</i> control, both 3 and 7 DPI. <i>Tlr4^−/−/Sigirr^−/−</i> mice showed a statistically significant increase, relative to control mice at 3 DPI only, but even at 3 DPI, were significantly less than either <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice. Statistical significance was determined using a two-way ANOVA and a Bonferroni post-test (NS p>0.05, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).</p

Immunofluorescence staining of <i>C. jejuni</i> cecal colonization <i>in vivo</i>.

<p>Formalin-fixed tissue sections of ceca obtained from <i>C. jejuni</i>-infected WT and <i>Sigirr^−/−</i> mice 7 DPI at ×200 magnification. Cell nuclei are stained with DAPI (blue), epithelial cells are outlined with antibodies specific to β-actin (green), and <i>C. jejuni</i> (red) are clearly visible around the edge of the lumen and into the cecal crypts.</p

Colonization and pathology of <i>Sigirr^−/−</i> and TLR-deficient mice by <i>C. jejuni ΔkpsM in vivo</i>.

<p>(A) H&E stained histological sections of ceca recovered from <i>Sigirr^−/−</i>, <i>Tlr2^−/−/Sigirr^−/−</i> and <i>Tlr4^−/−/Sigirr^−/−</i> mice, colonized with <i>C. jejuni ΔkpsM</i> 7 DPI, at 100× magnification. Very severe inflammation is evident in the <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice, however once again, no significant pathology was evident in the <i>Tlr4^−/−/Sigirr^−/−</i> mice. (B) Immunofluorescence of <i>Sigirr^−/−</i> mice infected by <i>C. jejuni ΔkpsM</i>, 7 DPI. Sections are stained for DAPI (blue), β-actin (green), and <i>C. jejuni</i> (red). <i>Sigirr^−/−</i> mice exhibit significant neutrophil infiltration, hyperplasia, and <i>C. jejuni ΔkpsM</i> is clearly visible in large masses within the cecal crypts. (C) Pathological scoring was done by two blinded observers, using H&E stained, formalin-fixed cecal tissue sections. Each condition represents a minimum of three separate experimental replicates, with 2–3 mice per experiment. Control mice were used as a reference and consisted of 3 uninfected mice, pre-treated with a single dose of 5 mg/100 µl vancomycin, and euthanized 3 days post-treatment. Only <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice at 7 DPI showed a significant increase in pathology (****p<0.0001), relative to the uninfected <i>Sigirr^−/−</i> control. The <i>Tlr2^−/−/Sigirr^−/−</i> mice also exhibited statistically significantly higher inflammation at 7 DPI relative to <i>Sigirr^−/−</i> mice also at 7 DPI (**p<0.001). In contrast, none of the mouse strains at 3 DPI showed any statistically significant increase in pathology, relative to control mice. Statistical significance was determined using a two-way ANOVA and a Bonferroni post-test.</p

Immunofluorescent staining of intracellular <i>C. jejuni in vivo</i>.

<p>(A) Intracellular <i>C. jejuni</i> are visible in the cecal epithelium. <i>C. jejuni</i> (red), are visible against the β-actin (green) and the nuclei (DAPI, blue) of the cecal epithelium of a <i>Sigirr^−/−</i> mouse, ×1000 magnification. (B) Confocal image of <i>C. jejuni</i> (red) present within epithelial cells of the colon of a <i>Sigirr^−/−</i> mouse, highlighted against the Cytokeratin 19 of the cytoskeleton (green) and the nuclei (blue), with the z-stack cross-section indicating the <i>C. jejuni</i> within the cell. (C) Cross-section of a Z-stack, of a colonic epithelial cell of a <i>Sigirr^−/−</i> mouse. The internalized <i>C. jejuni</i> (red) are clearly visible within the cytoplasm of the cell, as outlined by the β-actin (green) along the edge of the cell. (D) Internalized <i>C. jejuni</i> (red) co-localize with LAMP-1 positive (green) vesicles present within epithelial cells of a <i>Sigirr^−/−</i> mouse colon.</p

Colonization of WT and <i>Sigirr−/−</i> mice by <i>ΔkpsM</i> and <i>ΔflaA</i>.

<p>Colonization of WT and <i>Sigirr^−/−</i> mice by <i>ΔkpsM</i> (A) and <i>ΔflaA</i> (B) and their respective complemented strains (<i>ΔkpsM</i>+<i>kpsM</i> and <i>ΔflaA</i>+<i>flaA</i>), at both 3 and 7 DPI. The <i>ΔkpsM</i> mutant exhibited reduced colonization at 3 DPI only, while the <i>ΔflaA</i> mutant was unable to colonize at either 3 or 7 DPI. The complemented <i>ΔflaA+flaA</i> colonized at high numbers, similar to wild-type. Statistical significance was determined by a Mann-Whitney test, ***p<0.001. n = 7–10 mice for the <i>ΔkpsM</i> mutant and complement, n = 5–7 mice for the <i>ΔflaA</i> mutant and complement. (C) H&E stained histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔflaA</i> at ×100 magnification. No noticeable inflammation was evident in either WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔflaA</i>. (D) H&E stained histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔkpsM</i> 7 DPI. Upper panels are ×100 magnification, while lower panels are ×400 magnification. WT mice did not exhibit signs of inflammation when infected with <i>C. jejuni ΔkpsM</i>, however <i>Sigirr^−/−</i> mice exhibited signs of severe inflammation at 7 DPI.</p

Cytokine production in infected mice.

<p>(A–H) RT-qPCR conducted on RNA extracted from the ceca of uninfected control or infected mice. Controls are the pooled results of 9, vancomycin pre-treated, but uninfected mice, euthanized 3 days post-treatment. All infected mice represent the average results of 3 independent experiments, each of which include the pooled RNA of 2–3 mice, for 6–9 mice total for each mouse strain, euthanized either 3 or 7 DPI. Statistical significance was determined using a One way ANOVA with a Bonferroni post-test. * p<0.05 relative to WT (B6) or <i>Sigirr^−/−</i> uninfected control mice. ** p<0.05 relative to the infected WT (B6) mice euthanized on the same DPI in addition to the uninfected control mice.</p