Primary human choroidal melanocytes express functional Toll‐Like Receptors (TLRs)

Abstract

PurposeHuman choroidal melanocytes (HCMs) are an abundant heterogeneous, melanin‐containing population of cells within the vascular uveal tract. Toll‐like receptors (TLRs) are pattern recognition receptors involved in the innate immune response against phylogenetically conserved microbial components. Immune cells are established to express TLRs and more recent studies showed TLR expression on non‐immune cells including corneal and conjunctival epithelia, retinal microglia and RPE, iris pigment epithelium, and skin melanocytes. The expression of TLRs on uveal melanocytes however remains to be investigated.MethodsHCMs were isolated and cultured from donor human eyes. TLR1‐10 and MYD88 (a critical adaptor protein in the TLR signalling pathway) expression was investigated using RT‐PCR and confirmed by immunocytochemistry. The production of IL‐8 and CCL2 in response to TLR agonists was measured by ELISA.ResultsHCMs constitutively expressed TLR1‐6, TLR9, and MYD88. Stimulation of HCMs with bacterial‐derived TLR agonists [Pam3CSK4 (TLR1/2), HKLM (TLR2), LPS (TLR4), Flagellin (TLR5) and, FSL‐1 (TLR6/2)] and virus‐mimicking TLR agonists [Poly I:C (TLR3), Imiquimod (TLR7), ssRNA40 (TLR8) and ODN2006 (TLR9)] induced variable secretion of IL‐8 and CCL2 (MCP‐1). Control levels of IL8 (140 pg/ml) increased in response to Pam3CSK4 (2360 pg/ml), Poly I:C (3620 pg/ml), LPS (4100 pg/ml), Flagellin (1550 pg/ml) and FSL‐1 (2160 pg/ml). Virus‐mimicking agonists imiquimod, ssRNA40 and ODN2006, and intriguingly bacteria‐derived HKML, did not significantly affect secreted IL8. Secreted CCL2 showed a similar pattern of response following stimulation with TLR agonists. HCMs were immunoreactive for TLR2, TLR3 and TLR4 protein.ConclusionsThe findings suggest that HCMs are potentially involved in innate immune responses against bacterial and viral pathogens within the human choroid.