Transmembrane immune receptor CD300lf attenuates microglial inflammasome signaling in vitro

Purpose:
Neuroinflammation contributes critically to glaucoma pathogenesis and is thought to be driven in part by microglia, the resident immune cells of the central nervous system. Although microglia have been previously shown to influence disease progression, the mechanism by which these cells mediate neuroinflammation remains poorly understood. CD300lf is a transmembrane, lipid-sensing immune receptor expressed by microglia and macrophages that modulates immunometabolism, inflammatory phenotype and phagocytosis in these cells. CD300lf is upregulated by microglia in mouse glaucoma models. We hypothesize that CD300lf plays a critical role in regulating microglial proinflammatory response in glaucoma. We investigated the effect of genetic deletion of CD300lf on microglial phagocytic ability and response to inflammatory stimuli.

Methods :
Primary microglial cultures were obtained by isolating microglia from brains of C57BL/6J (wildtype) and CD300lf –/– mice. Inflammasome activation was evaluated in vitro following pre-stimulation with lipopolysaccharide (LPS) for either 6 or 24 hours, followed by treatment with either nigericin (2 hours) or ATP (30 min). Changes in gene expression were determined using qPCR. The ability of CD300lf –/– microglia to phagocytose fluorescently labeled apoptotic neurons (isolated primary retinal ganglion cells or the neuronal cell line SH-SY5Y) was evaluated using FACS

Results:
While both wildtype and CD300lf –/– microglia strongly responded to LPS, CD300lf –/– microglia showed a statistically significant increase in Il1β mRNA level compared to wildtype in response to LPS and ATP at the 6-hour timepoint in two independent experiments. There was no significant difference in Nlrp3, Il18 or Aim2 mRNA levels between the two groups. In the phagocytosis assay there was no difference in the phagocytic ability between CD300lf –/– and wildtype microglia.

Conclusions:
Loss of function in CD300lf did not directly affect the phagocytic ability of microglia, but it heightened their proinflammatory response to LPS and ATP in terms of Il1β production. Thus, CD300lf acts as a novel modulator of inflammasome signaling in microglia in vitro and its contribution to glaucoma pathogenesis merits further investigation in vivo.