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  • Soluble receptors have been shown


    Soluble receptors have been shown to be important and common regulators of many cytokines within the immune system, including IL-1, IL-6, IFNγ, TNF-α, GM-CSF, G-CSF and others (Fernandez-Botran, 1991, Heaney, Golde, 1998). We previously described a novel soluble receptor, sCSF-1R, that to date has only been found in teleost fish. In primary macrophage cultures, sCSF-1R was found in highest levels during periods of significant inhibition of macrophage proliferation and differentiation. In the senescent phase of goldfish in vitro macrophage development, increases in macrophage production of sCSF-1R paralleled the induction of apoptotic events (Barreda et al., 2005). Further, recombinant sCSF-1R inhibited macrophage proliferation pointing to a potential role in hematopoiesis control (Barreda et al., 2005). Aside from its direct effect on macrophages, our results in this study show that this novel soluble protein also contributes to the control of teleost fish inflammation by modulating neutrophil activity. We found that CXCL-8, a chemokine known to be involved in neutrophil recruiting, was greatly decreased at all concentrations of sCSF-1R. Similarly to what has been described in mammals (Harada et al., 1994), CXCL-8 may be the primary chemokine involved in neutrophil recruitment in goldfish. While CXCL-8 was greatly affected, we also found significant decreases in CCL-1, a recruiter of monocytes and lymphocytes. The mRNA AG-126 data presented in this study matched closely with our previously published results, where sCSF-1R treatment inhibited monocyte, macrophage, and lymphocyte infiltration (Rieger et al., 2013). The changes in chemokine expression described above paralleled a significant inhibition in the expression pro-inflammatory cytokines including IL-1β1 and IL-1β 2, IFNγ and IFNγrel, TNF-α2, IL-12p35 and CSF-1. All of these have been previously implicated in the regulation of goldfish macrophage pro-inflammatory responses, especially phagocytosis and the production of reactive intermediates (Grayfer, Belosevic, 2009, Grayfer et al, 2008, Grayfer et al, 2009, Grayfer et al, 2010). Notably, for TNFα2 and CSF-1 we identified a differential decrease in mRNA expression at the lowest sCSF-1R concentrations tested. It will be interesting to examine the impact of low doses of sCSF-1R across a panel of pro-inflammatory cytokines to assess whether this is a general phenomenon highlighting a differential level of sensitivity to sCSF-1R. When we studied canonical anti-inflammatory responses, we found further differences between responses to sCSF-1R and apoptotic cells. The most dramatic effect was seen on IL-10 expression. Unlike the pro-inflammatory cytokines, we found that an increase in IL-10 expression was limited to lower concentrations of sCSF-1R. Interestingly, our work did not show an increase in TGF-β following apoptotic cell exposure, even though this represents the primary mediator of anti-inflammatory responses following phagocytosis of apoptotic cells in mammals (Fadok et al., 1998). It is possible that sCSF-1R may exert differential responses at other concentrations for TGF-β and other anti-inflammatory cytokines, or that additional or different triggers may be required to induce production following exposure to apoptotic cells in teleost fish. Activation of neutrophil responses is highly dependent on the soluble factors that they are exposed to upon entering an inflammatory site. Thus, we examined if changes to complex in vivo milieu induced by apoptotic cell or sCSF-1R could affect neutrophil-specific inflammatory responses. We found that both the presence of sCSF-1R and apoptotic cells significantly reduced neutrophil phagocytosis. Finally, the changes to the inflammatory milieu induced by sCSF-1R and apoptotic cells significantly reduced the capacity of neutrophils to clear phagocytosed bacteria. This likely reflects the importance of a timely inflammation control response both for the effective clearance of attacking pathogens and the induction of tissue repair/homeostatic programs.