Colonic Immune Suppression, Barrier Dysfunction, and Dysbiosis by Gastrointestinal <i>Bacillus anthracis</i> Infection

<div><p>Gastrointestinal (GI) anthrax results from the ingestion of <i>Bacillus anthracis</i>. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated <i>B. anthracis</i> Sterne strain (pXO1⁺ pXO2⁻) spores that resulted in rapid animal death. <i>B. anthracis</i> Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only <i>B. anthracis</i>, but also of commensals. Disease progression significantly correlated with the deterioration of innate and T cell functions. Our studies provide critical immunologic and physiologic insights into the pathogenesis of GI anthrax infection, whereupon cleavage of mitogen-activated protein kinases (MAPKs) in immune cells may play a central role in promoting dysfunctional immune responses against this deadly pathogen.</p></div

Cleavage of MAPKs in DCs of Sterne-Infected A/J Mice.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and DCs analyzed at various time points. <b>A</b>. Gating strategy for the analysis of CD45⁺MHCII^hiCD11c⁺F4/80⁻CD11b⁺ colonic DCs. <b>B</b>. Cell surface expression of CD86, CD80, and B7-H1 by colonic DCs was analyzed by flow cytometry. Gray tinted line = isotype control; black line = PBS group; green line = day 1 Sterne-infected A/J mice; magenta line = day 3 Sterne-infected A/J mice; blue line = day 5 Sterne-infected mice. <b>C</b>. Activity of MAPKs in colonic DCs of infected versus uninfected mice was analyzed by flow cytometry. Gray tinted line = isotype control; black line = PBS group; blue line = day 5 Sterne-infected mice. <b>D</b>. Colonic LP cells were isolated from uninfected A/J mice and incubated with 1 MOI of <i>B. anthracis</i> spores for 1, 3, or 6 hours. Activity of p38 and Erk1/2 was subsequently analyzed in colonic DCs. Gray tinted line = isotype control; black line = PBS group; green line = 1 hour treatment; magenta line = 3 hour treatment; blue line = 6 hour treatment. Data represent observations from three independent experiments and are shown as mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001 compared with PBS.</p

T Cell Responses in Sterne-Infected A/J Mice.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and adaptive immune responses in the colon analyzed at various time points by flow cytometry. <b>A</b>. Gating strategy for the analysis of colonic T cells. <b>B</b>. Th1, Th17, and regulatory T cells responses were tested by flow cytometry. Representative plots indicate cytokine production of uninfected and day 5-infected mice. <b>C</b>. Surface expression of PD1 in colonic T cells. <b>D</b>. Gene expression profile of the distal colon of Sterne-infected A/J mice. Data represent observations from four independent experiments and are shown as mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001 compared with PBS.</p

GI Dysbiosis Subsequent to Sterne Infection.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and changes in microbiota composition during the course of Sterne infection were monitored. <b>A</b>. Unweighted UniFrac analyses were used to calculate distances between samples obtained from Sterne-infected A/J mice before infection and three days post-infection and three dimensional scatterplots were generated by using principal coordinate analysis (PCoA); n = 9 mice/group. <b>B</b>. Average abundance values of indicated phyla. <b>C</b>. Decreased microbial diversity, evenness, and species richness. Left: The Chao richness index was used as a measure of species richness. Middle: The Shannon diversity index was used to estimate microbial diversity for each group. Right: The species evenness index was calculated using the formula J’ = H’/H’_max, where H’ is the Shannon diversity index and H’_max is the maximal value of H’. Data are shown as mean +/− SEM. *P<0.05, ***P<0.001 compared with PBS-treated or day 0 mice. <b>D</b>. Bacteria genera most enriched or depleted in Sterne-infected mice at day 3 versus day 0, as measured by linear discriminant analysis (LDA). <b>E</b>. Reduced relative abundance of Enterobacteriaceae and <i>Bifidobacterium</i> in Sterne-infected A/J mice. <b>F</b>. Presence of Enterobacteriaceae in MLNs, spleens, and livers of Sterne-infected mice. <b>G</b>. Persistence of <i>B. anthracis</i> spores in the feces of Sterne-infected mice.</p

Suppression of Innate Immune Responses in Sterne-Infected A/J Mice.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and innate immune responses analyzed at various time points. <b>A</b>. DCs isolated from the colons of Sterne-infected A/J mice were analyzed by flow cytometry for the production of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6. Representative plots indicate cytokine production of uninfected and day 3 infected mice. Corresponding isotype controls were utilized for gating of intracellular cytokines. <b>B</b>. Gene expression profile of the distal colon of Sterne-infected A/J mice. Data represent observations from four independent experiments and are shown as mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001 compared with PBS.</p

Lethality and Systemic <i>B. anthracis</i> Sterne Dissemination in A/J Mice.

<p>A. A/J mice were orally gavaged with 10⁵, 10⁷, or 10⁹ spores of the Sterne strain of <i>B. anthracis</i>. Lethal infection was established within 3 days in A/J mice receiving 10⁹ spores; n = 10 mice/group (10⁵ and 10⁷), n = 20 mice/group (10⁹). Experiments were performed a minimum of three times. Statistical significance was calculated using the log-rank test. After 3 days of infection, A/J mice were sacrificed; both spores and vegetative bacilli (marked with *) were observed in the colon (<b>B</b>), MLNs (<b>C</b>), spleen (<b>D</b>, <b>E</b>), liver (<b>F</b>), kidneys (<b>G</b>), lungs (<b>H</b>), and in bronchoalveolar lavage (BAL) fluid (<b>I</b>). Bar = .</p

GI Epithelial Barrier Dysfunction Induced by Sterne Infection.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and intestinal barrier integrity was analyzed <i>ex vivo</i> three days post-infection; n = 5 mice/group. TEER (<b>A</b>), trans-epithelial conductance (<b>B</b>), and short-circuit current (<b>C</b>) of Sterne-infected versus uninfected A/J mice. <b>D and E</b>. Delta current (Δ<i>I_SC</i>) before and after cholinergic (<b>D</b>) and histamine (<b>E</b>) challenges. <b>F</b>. Post-challenge secretory current of Sterne-infected versus uninfected A/J mice. Data are shown as mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001 compared with PBS. <b>G</b>. Colonoscopies were performed in groups of uninfected and 10⁹ Sterne spores-infected A/J mice three days post infection with a Multi-Purpose Rigid Telescope attached to a TELE PACK X. <b>H</b>. Gross hemorrhage in the small intestines<b>. </b><b>I and J.</b> Hemorrhagic lesions in the colon (<b>I</b>) and small intestine (<b>J</b>) of Sterne-infected A/J mice. Bar = 200 µm.</p