The C5a receptor antagonist PMX205 ameliorates experimentally induced colitis associated with increased IL-4 and IL-10

Abstract

Background and purpose: Anti-complement therapies have not been advanced for treating the inflammatory bowel diseases (IBDs) despite a growing body of evidence that blocking C5a protects against induced colitis in rodents. The purpose of this study was to further build on this evidence by examining the efficacy, mechanism and specificity of a potent, non-competitive and orally active C5a receptor (CD88) antagonist, PMX205, in the dextran sulphate sodium (DSS) model of murine innate colitis.

Experimental approach: Mice with DSS added to their drinking water were orally administered 100 or 200 μg day(-1) PMX205 in prophylactic and therapeutic regimens. Clinical illness, colon histology and local generation of inflammatory mediators were measured to evaluate the impact of PMX205 on disease.

Key results: PMX205 significantly prevented DSS-induced colon inflammation in both regimens, associated with lower pro-inflammatory cytokine production and nitrotyrosine staining in colon sections. Additionally, the levels of anti-inflammatory cytokines IL-4 and IL-10 were increased. PMX205 had no significant effect on C5a levels. The beneficial effect of PMX205 was seen in two strains of mice of differing sensitivities to DSS inflammation, but was inactive in mice lacking CD88.

Conclusions and implications: Pharmacological inhibition of C5a activity by PMX205 is efficacious in preventing DSS-induced colitis, providing further evidence that targeting CD88 in IBD patients could be a valuable therapeutic option.

Figure 1

DSS-induced local generation of C3a and C5a is not altered by PMX205. BALB/c mice were fed 5% DSS in water and treated with either water (control) or PMX205 (100 or 200 μg per mouse) in a prophylactic regimen until day 6. Untreated mice were kept on facility water. On the day of killing, the colon was excised and explant supernatants were analysed for C3a (A) and C5a (B) by ELISA. Values are shown as mean concentrations ± SEM (n = 5–11 mice per group). All DSS-treated groups are significantly different from the untreated group.

Figure 2

Effect of PMX205 on DSS-induced body weight loss, clinical disease and colon length in BALB/c mice. BALB/c mice had 5% DSS added to their water for 5 days. PMX205 (100 or 200 μg/mouse) or water (control) was administered orally beginning 1 day prior (Day 0) to the DSS start. The untreated group was kept on facility water until day 7. (A) Each animal's weight was measured daily and is expressed as the percentage of their weight on day 1. Shown are the averages ± SEM (n = 5–13 mice per group). (B) Clinical illness scores were derived from combinations of weight loss, stool consistency and presence of blood in stool. (C) Colon lengths from the groups were measured after killing. Results are expressed as mean colon length ± SEM (n = 5–13 per group). *P < 0.05, **P < 0.01 and ***P < 0.001 versus controls. Although different from the DSS control group, the PMX205 group also remained statistically different from the untreated group.

Figure 3

PMX205 attenuates DSS-induced histopathological features in BALB/c mice. (A) Representative figures of the colon prepared from (a,d) untreated, (b,e) control and (c,f) PMX205 groups. In (b,c) ovals identify the ulcers along the length of the colon. Compared with the control group, PMX205-treated mice typically showed fewer and shorter ulcers, less crypt damage, fewer mice with oedema and less of the colon was infiltrated. (B) The inflammation score was determined from these criteria by an investigator blinded to the treatment. Each dot is the data from a single mouse and the line represents the median of the inflammation scores. **P < 0.01 versus controls.

Figure 4

Effect of PMX205 on DSS-induced granulocyte infiltration and myeloperoxidase: Representative examples of immunohistochemical staining for (A) Ly6G and (B) F4/80. Quantification of stained cell numbers of (C) neutrophils and (D) macrophages in the mucosa and submucosa of DSS groups. Data are shown as mean ± SEM (n = 5–8/group). Original magnification: 400×. (E) MPO levels were not significantly different between the DSS groups although both groups were different from untreated values. Values shown are mean O.D. ± SEM (n = 5/group). *P < 0.05. An isotype control antibody did not show any visible staining (data not shown).

Figure 5

PMX205 prevents the production of pro-inflammatory cytokines and induces the production of IL-4 and IL-10. Colon culture supernatants from untreated, PMX205 or water-treated DSS inflamed mice were analysed for the production of (A) IL-6, (B) TNF, (C) IL-1β, (D) IL-12, (E) CXCL2, (F) IL-17, (G) IL-4 and (H) IL-10. Values shown are mean concentrations ± SEM (n = 5–10 mice per group). In panels A through F, the cytokine levels of the DSS control colons are significantly different from untreated levels but not in G and H, shown in the figure. *P < 0.05, **P < 0.01 PMX205 versus DSS controls.

Figure 6

Effect of PMX205 on NO activity estimated by NT staining of colon sections. (A) Representative examples of immunohistochemical staining for NT. (B) Quantitation of the staining signal identifies a significant reduction in the intensity of NT in the PMX205 group. An isotype control antibody did not show any visible staining (data not shown). *P < 0.05 versus control.

Figure 7

PMX205 did not abrogate DSS-induced damage in mice lacking CD88. PMX205 was used in the prophylactic regime in CD88^−/− mice to test the drug specificity for CD88. (A) PMX205 had no protective effect on colon shortening due to the DSS. Shown are the mean colon lengths ± SEM (n = 5–7 mice per group). (B) Representative sections of the colon of inflamed CD88^−/− mice. Red ovals identify ulcers. (C) Inflammation scores derived from the histology; each dot represents a single mouse and the line represents the median score.

Figure 8

PMX205 ameliorates DSS induced body weight loss, colon shortening and histopathological changes in C57BL/6 mice. C57BL/6 mice had 3% DSS added to their water for 5 days. Untreated mice were kept on facility water until day 7. PMX205 or water (control) was administered orally beginning 1 day prior to the DSS start (day 0). PMX205 significantly prevented the (A) body weight and (B) colon shortening due to the DSS exposure. Results are presented as mean ± SEM (n = 5–14 mice per group). (C) Haematoxylin and eosin-stained colons from (a,d) untreated, (b,e) control or (c,f) PMX205-treated group. (D) Inflammation scores, where each dot represents a single mouse and the line represents median score. *P < 0.05 and **P < 0.01.

Figure 9

Effects of therapeutic PMX205 on measures of DSS-induced colitis in BALB/c mice. Mice were fed 5% DSS in drinking water from day 1 until day 6. PMX205 or water (control) was orally administered starting from day 3 through day 6. PMX205 significantly reduced measures of colitis including (A) body weight loss, (B) colon shortening, (C) clinical illness and (D) colon inflammation scores. Results are presented as mean ± SEM (n = 6–8 mice/group). *P < 0.05 and ***P < 0.001 versus DSS controls.