Depletion of enteric bacteria diminishes leukocyte infiltration following doxorubicin-induced small intestinal damage in mice

Figures

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  Fig 1. Doxorubicin (Doxo) induces weight loss in non-antibiotic treated (NoAbx) mice, but does not induce weight loss in antibiotic-treated (Abx) mice. NoAbx and Abx were treated with a single dose of intraperitoneal Doxo at 20 mg/kg. Percent body weight loss at 6 hours, 24 hours, 3 days and 5 days was calculated from weight at injection. n = 3–5 per time point per group. * = values significantly different from respective control; P<0.05

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  Fig 2. Doxorubicin (Doxo) induces equivalent apoptosis in antibiotic-treated (Abx) and non-antibiotic treated (NoAbx) mice. A. Quantification of the number of apoptotic cells per crypt in Abx and NoAbx jejunal tissue from control mice and 6 hours after Doxo treatment. n = 3–4 per time point per group. * = values significantly different from respective control; P<0.05. B. Immunofluorescence staining at 64X demonstrating active caspase 3-positive cells (pink) in Ab and NoAbx jejunal tissue from control mice and 6 hours after Doxo treatment.

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  Fig 3. Doxorubicin (Doxo) induces crypt loss followed by crypt deepening and proliferation in non-antibiotic treated (NoAbx) mice, but not in antibiotic-treated (Abx) mice. A. Number of crypts was quantified in control mice and 5 days following treatment with Doxo. Results expressed as total number of crypts per 1000 μm (left) and total number of crypts per cross-section (right). B. Crypt depth (μm) was quantified in control mice and 5 days following treatment with Doxo. C. H&E staining of representative cross-sections at 64X. Yellow arrowheads denote individual crypts. D. Proliferation was evaluated by quantification of pHH3+ cells in control mice and 5 days following treatment with Doxo. E. IHC staining of representative cross-sections at 64X. Brown cells denote pHH3+ cells. n = 3–5 per time point per group. * = values significantly different from respective control; P<0.05. # = values significantly different between groups (NoAbx & Abx) within a specific time point; P<0.05.

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  Fig 4. Doxorubicin (Doxo) does not induce an increase in inflammatory cytokines but does induce an increase in immune-modulating chemokines in non-antibiotic treated (NoAbx) mice; this increase is dampened in antibiotic-treated (Abx) mice. A. Fold change in mRNA of TNFα, IL1β, and IL10 in NoAbx jejunal tissue from control mice, 6 hours and 5 days after Doxo treatment. n = 5–8 mice per time point. B. Fold change in mRNA of CCL2, CCL7, and KC in Abx and NoAbx jejunal tissue from control mice, 6 hours and 5 days after Doxo treatment. n = 3–5 mice per time point per group. * = values significantly different from respective control; P<0.05. # = values significantly different between groups (NoAbx & Abx) within a specific time point; P<0.05.

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  Fig 5. Doxorubicin (Doxo) induces an influx of macrophages and neutrophils in non-antibiotic treated (NoAbx) mice, but not in antibiotic-treated (Abx) mice. A. Quantification of the number of macrophages (F4/80+) per crypt unit in Abx and NoAbx jejunal tissue from control mice, 24 hours and 5 days after Doxo treatment. B. IHC staining of representative cross-sections with brown cells denoting F4/80+ cells. C. Quantification of the number of neutrophils (anti-neut+) per crypt unit in Abx and NoAbx jejunal tissue from control mice, 24 hours and 5 days after Doxo treatment. D. IHC staining of representative cross-sections with brown cells denoting anti-neut+ cells. n = 3–4 per time point per group. * = values significantly different from respective control; P<0.05. # = values significantly different between groups (NoAbx & Abx) within a specific time point; P<0.05.

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  Fig 6. Doxorubicin (Doxo) induces an influx of macrophages and neutrophils in conventionally-raised (CONV) mice, but not in germ free (GF) mice. A. Quantification of the number of macrophages (F4/80+) per crypt unit in CONV and GF jejunal tissue from control mice, 6 hours, 3 days and 5 days after Doxo treatment. B. Quantification of the number of neutrophils (anti-neut+) per crypt unit in CONV and GF jejunal tissue from control mice, 6 hours, 3 days and 5 days after Doxo treatment. n = 3 per time point per group. * = values significantly different from respective control; P<0.05. # = values significantly different between groups (CONV & GF) within a specific time point; P<0.05.