Plasmacytoid Dendritic Cells Play a Key Role in the Efficacy of GM-CSF in Murine IBD Models

Abstract

Purpose: Crohn's disease is a chronic inflammatory bowel disorder associated with a loss of tolerance to commensal gut flora. A phase II clinical trial demonstrated that Sargramostim (recombinant hGM-CSF) was effective in the treatment of patients with moderate-to-severely active Crohn's disease (NEJM 2005; 352:2193–2201). We previously demonstrated that pegylated murine-GM-CSF (pGM) also abrogates colitis in mouse models; effects were blocked by a novel mAb 440c that targets the Siglec-H receptor, unique to plasmacytoid dendritic cells (pDCs). To establish the MOA of GM-CSF in the treatment of Crohn's disease, we investigated GM-CSF effects on 440c+ pDCs, their location in the mucosa, mAb 440c effects on cytokine expression in DSS colitis, and the dependence on adaptive immune elements. Methods: Mice were treated IP with pGM (5ug/d for 5 days), saline, or 22E9 fxn-blocking anti-GM-CSF (200ug) for five days. Splenic cells were analyzed by flow cytometry (CD11c+, 440c+, and B220+ markers). In situ localization was performed by tissue immunohistochemistry (IHC) using mAb 440c. DSS colitis models were performed in Balb/c mice, or RAG1-/- mice, lacking B and T cells, using DSS for 7 days. Groups were treated with PEG-rm-GM-CSF and/or mAb 440c to probe the involvement of the pDC in the response to GM-CSF. Disease activity score, histopathological score, and cytokine expression by real time RT-PCR and protein-arrays were determined. Results: GM-CSF was a potent regulator of the 440c+ pDC (or B220+), with decreases evident in 22E9 treated animals and increases following pGM treatment. pDCs were easily localized by IHC to the subepithelial region of the small intestine and colon. pGM significantly reduced DSS-colitis associated disease activity. However, the administration of mAb 440c blocked this protective effect. Significant decreases in TNFa and IL-1b expression by pGM were blocked by mAb 440c treatment; data further confirmed by protein-array. pGM was therapeutic in RAG1-/- model mice, demonstrating independence from adaptive immune elements. Conclusions: These data suggest that the pDC may play a central role in GM-CSF's therapeutic effects in IBD, and further support a key regulatory role for this small cell population in the mucosal immune response.