In vivo physiological and transcriptional profiling reveals host responses to Clostridium difficile toxin A and toxin B

Abstract

Toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile cause gross pathological changes (e.g., inflammation, secretion, and diarrhea) in the infected host, yet the molecular and cellular pathways leading to observed host responses are poorly understood. To address this gap, we evaluated the effects of single doses of TcdA and/or TcdB injected into the ceca of mice, and several endpoints were analyzed, including tissue pathology, neutrophil infiltration, epithelial-layer gene expression, chemokine levels, and blood cell counts, 2, 6, and 16 h after injection. In addition to confirming TcdA's gross pathological effects, we found that both TcdA and TcdB resulted in neutrophil infiltration. Bioinformatics analyses identified altered expression of genes associated with the metabolism of lipids, fatty acids, and detoxification; small GTPase activity; and immune function and inflammation. Further analysis revealed transient expression of several chemokines (e.g., Cxcl1 and Cxcl2). Antibody neutralization of CXCL1 and CXCL2 did not affect TcdA-induced local pathology or neutrophil infiltration, but it did decrease the peripheral blood neutrophil count. Additionally, low serum levels of CXCL1 and CXCL2 corresponded with greater survival. Although TcdA induced more pronounced transcriptional changes than TcdB and the upregulated chemokine expression was unique to TcdA, the overall transcriptional responses to TcdA and TcdB were strongly correlated, supporting differences primarily in timing and potency rather than differences in the type of intracellular host response. In addition, the transcriptional data revealed novel toxin effects (e.g., altered expression of GTPase-associated and metabolic genes) underlying observed physiological responses to C. difficile toxins.

Fig 1

Flow diagram outlining the experimental methods and analyses in this study.

Fig 2

Physiological and gene expression changes after toxin injection. Panels A, B, and C include data combined from four independent experiments with 7, 39, 36, and 12 mice, respectively. In total, 10 of 94 mice did not survive until the experimental endpoint: one TcdA-treated mouse did not survive to 2 h, six mice did not survive to 6 h (1 TcdA, 2 TcdA+B, 1 TcdB, and 2 sham-challenged mice), and six mice did not survive to 16 h (3 TcdB and 3 TcdA mice; see Table S1 in the supplemental material). The data points displayed in the figure were used for each statistical test. The horizontal lines above the bar charts which connect two sample groups indicate a two-sample statistical test. The P values for these tests are indicated beside the lines. (A) Representative examples of H&E-stained cecal tissue sections from the 11 indicated sample groups. (B) Total histopathology score (see Materials and Methods) from cecal tissue sections. Except for the two mice injected with TcdA+B (two mice not used for microarrays), histopathology scores were not measured for mice that did not survive. Since two of three mice injected with TcdA+B did not survive to 6 h in our first experiment, we dedicated more mice for TcdA and TcdB at 16 h so that no samples were obtained for TcdA+B at 16 h. All subsequent experiments also excluded the 16-h time point for injection of TcdA+B. (C) The number of cells within the mucosa and immediate submucosa which were positive for MPO after immunohistochemical staining. *, P = 0.055 by the two-sided t test. (D) Venn diagrams show the overlap of which microarray probe sets are differentially expressed (comparing toxin-challenged mice to sham-challenged mice using a cutoff of q < 0.01; see Materials and Methods). All microarray probes are annotated into 45,501 probe sets, each of which represents the expression of one gene or multiple similarly related genes. Since only one microarray was used for TcdA+B at 6 h, statistical tests could not be used to determine differentially expressed genes for that sample group. (E) All probe sets which were differentially expressed for at least one time point were included in the heat map. The Pearson correlation coefficients below the heat map are generated by comparing the log fold changes between each sample group. The dendrogram above the heat map is a hierarchical clustering of the sample groups, using the correlation coefficients as the distance metric.

Fig 3

Biological functions associated with gene expression changes. The expression data were generated from the mice depicted in Fig. 2B. (A) The fold changes of differentially expressed GTPase and GTPase-binding genes. Only genes with greater than a 2-fold change in expression are shown. (B) Fold changes as described for panel A but instead showing genes associated with metabolic functions associated with gene expression changes. (C) Expression changes and clustering of genes annotated as being associated with immune regulation or inflammation. Genes were clustered based on expression changes 6 and 16 h after TcdA injection. In the scatterplots, black circles indicate genes with low expression changes; these genes are not included in the line plots.

Fig 4

Antibody neutralization of CXCL1 and CXCL2. In each panel, the four sample groups are defined by two binary factors: (i) TcdA injection or sham injection and (ii) pretreatment with isotype antibodies or anti-CXCL1 and anti-CXCL2 antibodies. The data in all panels are combined from two independent experiments, one with 24 mice and another with 14 mice (see Table S2 in the supplemental material). Missing values in panels A and B are due to the limited volume of blood that could be drawn from some mice. The data points displayed in the figure were used for each statistical test. Statistical tests are indicated with horizontal lines as described in the legend to Fig. 2. (A) Concentration of CXCL1 and CXCL2 in the sera of mice 6 h after cecal injection of TcdA. (B) Concentration of neutrophils in blood obtained by cardiac puncture. *, P = 0.057 by the Mann-Whitney U test. Using this nonparametric, two-sided test with three samples in one group and four in the other, the minimum possible P value is 0.057. P < 0.02 by the two-sided t test. (C) Total histopathology score (see Materials and Methods) from cecal tissue sections. (D) Survival of mice after cecal injection. Mice were monitored so that moribund mice were sacrificed and are counted as having not survived (nonsurvivors).