Figure 1.
DSS-induced colitis in Wt and KO mice.
a) Change in body weight of water- or DSS-challenged Wt and KO mice over 9 days of treatment. Data is expressed as the percentage of the original weight prior to treatment. ***p≤0.001 compared to Wt+DSS group. ***p≤0.001 compared to KO and Wt+DSS group. b) Corresponding clinical scores of Wt and KO mice over 9 days of DSS challenge. Clinical scores were generated by evaluating various presentations typically observed in the DSS colitis model, including reduced mobility, vocalization, and group interactions. *p≤0.05 and ***p≤0.001 compared to Wt+DSS mice. c) Feces of Wt and KO mice were analyzed and scored for fecal consistency, hematochezia, and rectal bleeding during the period of DSS or water challenge. d) Colon lengths as measured from the anus to the caecum. *p≤0.05 and ***p≤0.001. e) Weight of spleen on day 9 expressed as a ratio against original body weight prior to DSS or water challenge. f) Analysis of CD31 immunohistochemical staining of the transverse and descending colon in Wt and KO mice after 9 days of DSS challenge. l) Analysis of Ki67 immunohistochemical staining in the transverse and descending colon of unchallenged or DSS-challenged Wt and KO mice after 9 days. Data are represented in number of positive pixels per mm.
Figure 2.
Histopathological analysis and neutrophil enumeration of the colon in Wt and KO mice after 9 days of DSS challenge.
Sections of the transverse colon in a) control Wt mice, b) control KO mice, c) DSS-challenged Wt mice and d) DSS-challenged KO mice. DSS-challenged Wt mice had more cellular infiltration (N) and epithelial damage (U) than KO mice. New vascularization was observed in the submucosa and serosa layers (V). DSS-challenged KO mice exhibited less pathology as seen by relatively intact epithelium (U) and reduced cellular infiltration (N). e) Histopathological scores averaged from the transverse and descending colon in DSS-challenged and control Wt and KO groups. Histopathology was scored from 0 (no pathology), 5 (moderate pathology) to 10 (extreme pathology). *p≤0.05 and ***p≤0.001. f) Representative labelling of Gr-1+ cells in the f) control Wt mice, g) control KO mice, h) DSS-challenged Wt mice and i) DSS-challenged KO mice. There are sparsely scattered Gr-1+ neutrophils in unchallenged control Wt and KO mice. DSS-challenged Wt mice had more infiltrating neutrophils (N), particularly in the submucosa, compared to both untreated animals and DSS-challenged KO mice. j) Enumeration of immunohistochemically labelled Gr-1+ cells in the colon of both untreated animals and DSS-challenged mice. Data are represented as the number of cells per mm. *p≤0.05 and ***p≤0.001.
Figure 3.
Cytokine and chemokine expression in the colon following 9 days of DSS challenge.
a) IL-6, b) TNF-α, c) IFN-γ, d) MCP-1, e) IL-17a, f) IL-4, g) IL-10 and h) GM-CSF concentrations/cell counts in the colon of untreated and DSS-challenged Wt and KO mice after 9 days. Values are standardized against the protein concentration of colon homogenate supernatants. Assay was performed once and data are represented in pg/ml; *p≤0.05, **p≤0.01 and ***p≤0.001.
Figure 4.
Flow cytometric analysis of leukocytes in the colon of Wt and KO mice after DSS.
a) CD45+ leukocytes (CD45+), b) CD4+ T cells (CD45+ CD3+ CD4+), c) Ly6G+ neutrophils (CD45+ Ly6G+ Ly6C+ F4/80−) and d) F4/80+ macrophages (CD45+ F4/80+) in the colon following 9 days of either water or DSS-challenge. *p≤0.05.