Reactivity of monocyte-derived macrophages from patients with Crohn's disease

Abstract

Introduction: While usually seen as an autoimmune disease, the role of bacteria and pathogen-sensing by antigen presenting cells in the intestinal mucosa in Crohn's disease (CD) is becoming more apparent. The loss of integrity of the intestinal monolayer may allow bacterial penetration and trigger inflammation via interaction with tissue macrophages. Macrophages in the gut are solely derived from monocyte precursors in peripheral blood. They become resident (Type 2) macrophages upon entry into the mucosal environment. However, in CD, monocyte-derived macrophages (MDM) display Type 1 characteristics of pro-inflammatory cytokine production and activation. Here, we investigate macrophage interaction with enterocytes in the presence of pro-inflammatory signals in the gut. Methods: An “inverted” polarized monolayer of Caco-2 (human enterocyte) cells was established on membranes (Fig. 1). Monocytes were isolated from peripheral blood of patients with CD, ulcerative colitis (UC) or healthy controls (HC) and co-cultured with Caco-2 cells on the basolateral side of the membrane. We examined the effect of the monocytes on Caco-2 polarity, monocyte reactivity to enterocytes and their reactivity to IFN-γ, TNF-α, and LPS from enterotoxigenic E. coli. Results: Monocytes grown in conditioned media from Caco-2 cells showed morphologic changes of differentiation, including giant cell formation, without the need for direct cell-to-cell contact (Fig. 2). Monocytes from CD patients, but not HC or UC, reduced the TER of monolayers, an indication of monolayer integrity (Fig. 3A). Examining the membranes by IF suggested that monocytes were found at the edges of the disrupted monolayer (Fig. 3B). Macrophages from patients with CD showed a trend to more IFN-γ expression than macrophages from UC and HC. Intracellular cytokine staining of monocytes co-cultured with epithelial cells (polarized and non-polarized) showed no significant differences between markers of activation such as surface expression of CD69, or intracellular IFN-γ, or TNF-α. Macrophages from CD patients co-cultured with polarized Caco- 2 cells produced IL10, an anti-inflammatory cytokine, with little pro-inflammatory IL6 or IL8 produced in response to LPS and TNF-α (Fig. 4). Conclusion: Monocyte-derived macrophages from peripheral blood interact with enterocytes in a context-specific manner. Where there is a lack of polarity of the epithelial layer, it appears that the macrophages may attempt to support development of monolayer integrity. In the presence of pro-inflammatory signal therefore, they react in response to both their preset parameters and the signals they encounter in the gut. In patients with CD, there is evidence here that some degree of pre-programming influences their behaviour in this simple model.