Modulation of macrophage function and immune response by a helminth-derived cysteine protease

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Helminth-derived excretory/secretory (ES) products have been demonstrated to mediate the anti-inflammatory/regulatory environment associated with helminth infection (for a review see Allen et al. 2011). The ES products of helminths have been exploited for therapeutic benefit in both murine and human models of autoimmune diseases (Zaccone et al. 2003; Zheng et al. 2008; Motomura et al. 2009; Ruyssers et al. 2009; Johnston et al. 2010; Cancado et al. 2011; Carranza et al. 2012; Kuijk et al. 2012). In our laboratory, the ES products of the liver fluke trematode, Fasciola hepatica, have been shown to prevent autoimmune type 1 diabetes in a murine model (Lund et al. in preparation). Disease prevention was associated with the initiation and perpetuation of anti-inflammatory/regulatory immune responses, including the generation of alternatively activated macrophages, regulatory T cells and regulatory B cell populations (Lund et al. in preparation). Nevertheless, the individual molecular components within the ES responsible for these phenomenon are unknown. Therefore, HPLC fractionation of the ES products of Fasciola hepatica was undertaken. This revealed a number of components with immune-modulatory effects. One of these Fasciola hepatica products is a cysteine protease, cathepsin L1 (FhCL1), and in fact it comprises a large proportion of the total ES products. In mice, FhCL1 suppresses pro-inflammatory immune responses through cleavage of toll-like receptor (TLR)-3, resulting in modulation of cell signalling in peritoneal macrophages (Donnelly et al. 2010). This thesis therefore examines the effect of FhCL1 in human monocyte-derived macrophages. FhCL1 was shown to enhance expression of pro-inflammatory cytokines IL-6 and IL-8 in response to lipopolysaccharide. This was associated with the up-regulation of surface CD14, and the activation of TLR4 cell signalling via both the myeloid differentiation primary response gene 88 (MyD88)-dependent and toll-interleukin-1 receptor-domain-containing adaptor-inducing interferon-β (TRIF)-dependent signalling pathways. Furthermore, expression of IL-10 and co-stimulatory molecule CD86 was down-regulated in FhCL1-treated human monocyte-derived macrophages, and this was attributed to suppression of late endosomal TRIF-dependent signalling, with down-regulation of TRAF3. Although, FhCL1 modulated TLR signalling in human and murine macrophages, and suppressed TRIF-dependent signalling in both human and mouse macrophages, FhCL1 enhanced pro-inflammatory cytokine expression in human monocyte-derived macrophages. Therefore, FhCL1 modulates immune responses in human monocyte-derived macrophages, albeit differently from murine peritoneal macrophages. Furthermore, while FhCL1 degraded TLR3 in murine peritoneal macrophages, FhCL1 had no effect on TLR3 or TLR4 expression or localisation in human monocyte-derived macrophages. However, treatment with FhCL1 was shown to suppress the uptake of lipopolysaccharide (LPS) by human macrophages, which appeared to correlate with altered α-tubulin localisation. Thus suppressed uptake of LPS correlates with the suppression of TRIF-dependent late endosomal signalling. Nanotubes are cellular protrusions which connect cells and are utilised for the transport of cellular components between cells (reviewed in Gerdes et al. 2008; and Gurke et al. 2008). An incidental finding of this study was the observation of nanotubes connecting monocyte-derived macrophages in culture, and the documented trafficking of TLR4 between human macrophages, through these nanotubes. Interestingly, LPS stimulation enhanced the movement of TLR4 into nanotubes, but this was partially suppressed by FhCL1.
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