Abstract
Purpose :
Microglia are the innate immune cells of the retina. In response to photoreceptor damage microglia become activated and migrate to the outer retina where they clear cellular debris by phagocytosis. Phagocytosis can be facilitated by TAM family receptor-tyrosine kinases (Tyro3, Axl, Mertk), using bridging ligands such as Protein-S (ProS) to bind phosphatidylserine exposed on the surface of apoptotic cell bodies and photoreceptor outer segments (OS). We hypothesized that Mertk-mediated phagocytosis of OS limits the inflammatory response of microglia.
Methods :
To obtain a microglial cell model with endogenous expression of Mertk, microglial cells were isolated and cultured from brain of the Immortomouse®, which harbors a transgene encoding the SV40 mutant temperature-sensitive tsA58 tumor antigen. A clonal cell line, Immg4, was isolated and characterized. Western blotting and immunoprecipitation were used to measure Mertk protein expression and activating phosphorylation. For phagocytosis experiments, Immg4 cells were switched to serum-free medium and pretreated with human ProS, bovine OS, or a combination of the two, before being treated with lipopolysaccharide (LPS), a toll-like receptor-4 (TLR4) agonist. QRT-PCR was used to determine expression of mRNAs corresponding to pro- and anti-inflammatory factors.
Results :
Immg4 cells exhibited a ramified morphology and expressed markers characteristic of microglia, including: F4/80, CD45, CD68, CD11b, CD115, Iba-1, CD200R, TREM-2, CX3CR1 and Mertk. They became indefinitely quiescent when placed at non-permissive temperature (39°C), coinciding with greater ramification and increased expression of microglial markers and the differentiation factor PU.1. Immg4 exhibited efficient phagocytosis of OS that was dependent upon added ProS. Treatment of Immg4 cells with ProS plus OS caused phosphorylation of Mertk, indicating initiation of Mertk signaling. LPS significantly enhanced IL-1beta, TNF-alpha, and SOCS3 mRNA expression, which was abrogated by pretreatment with ProS plus OS, but not with ProS or OS alone.
Conclusions :
Immg4 cells represent a useful model for studying microglial function. ProS plus OS effectively inhibited inflammatory cytokine expression in response to a TLR4 ligand, suggesting that Mertk-mediated phagocytosis promotes a reparative phenotype in these cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.