| Popis: |
Originally described by Uhlig et. al., the anti-CD40 agonist acute model of gut inflammation is a useful tool for interrogating myeloid-driven gut inflammation. Previous studies have shown that this model is sensitive to intervention with anti-TNFα, anti-IFNγ and anti-IL12/ 23p40. Here we present data that extend previous findings in this model and demonstrate an anti-CD40 dose dependent severity of disease pathogenesis as well as dose response curves for treatment with anti-TNFα and anti-IL12/23p40. In addition, we have identified both circulating and fecal biomarkers of disease. Methods: RAG2 deficient mice on the C57BL/6 background were injected i.p. with a single dose of anti-CD40 agonist antibody (FGK45) on day 0 and monitored daily for body weight loss until day 7. Prophylactic treatment with either anti-TNFα or anti-IL12/23p40 was performed using i.p. injections on day -1 and day 2 prior to sacrifice at day 7. Spleens were collected and weights recorded, plasma and feces were also collected for drug exposure levels and biomarker analysis. The colon was removed, flushed with PBS and buffered formalin prior to histological sectioning and scoring. Results: Mice treated with anti-CD40 (single dose 1.25-20mpk) exhibited a dose-dependent increase in splenomegaly, colonic inflammation and liver pathology with an induction ED50 of 2.5mpk. In addition, plasma levels of CXCL-10, MCP-1, MIG and other inflammatory mediators were also increased dose-dependently with CXCL-10 levels highly correlating with histology scores (r2 = 0.85). Fecal calprotectin (S100A8-S100A9 dimer), a biomarker routinely used in the clinic, also increased dose-dependently and correlated well with histologic scores (r2 = 0.72 and 0.68 respectively). As significant liver necrosis was observed at doses of anti-CD40 above 5mpk, this dose was selected for intervention studies. Prophylactic treatment with either anti-IL12/23p40 or anti-TNFα attenuated colonic pathology in a dose-dependent manner at day 7 of disease with an EC50 of 100 μg/mL for anti-p40. Therapeutic intervention (i.e. a single injection given at day 3 of disease) with anti-TNFα also significantly reduced colon histopathology scores while antiIL-12/23p40 only showed therapeutic significance at the highest dose (EC50 of 7 and 1.3 μg/mL respectively). Reduced colonic inflammation in all cases was accompanied by a reduction in circulating CXCL-10 levels. In summary, we have shown that the anti-CD40 acute model of gut inflammation is a useful drug discovery model for interrogating myeloid mechanisms involved in colitis and have identified circulating CXCL-10 and fecal calprotectin as biomarkers related to both pathogenesis and intervention in this model. We have also generated dose-efficacy relationships for clinically validated therapies such as anti-TNF and anti-IL12/23p40. |
