The role of netrin-1 in the mouse cornea during Aspergillus fumigatus infection

Purpose: To explore the effects of netrin-1 on inflammation in Aspergillus fumigatus-infected mouse corneas and on proliferation and migration in human corneal epithelial cells (HCECs).

Methods: Netrin-1 and the receptor A2BAR were detected in normal and infected corneas from C57BL/6 mice and RAW 264.7 cells. The mice were injected subconjunctivally with recombinant netrin-1. The severity of the disease was determined by clinical scores, photography with a slit lamp, RT-PCR, western blotting, myeloperoxidase (MPO) assays and immunofluorescence staining of polymorphonuclear neutrophilic leukocytes (PMNs). The effects of netrin-1 on RAW 264.7 cells in vitro were determined by RT-PCR. The role of A2BAR was demonstrated in vivo by detecting the expression of IL-1β, TNF-α, and IL-10 in corneas pretreated subconjunctivally with an A2BAR antagonist (PSB1115). RAW 264.7 cells were stimulated with Aspergillus fumigatus (A. fumigates) and netrin-1 with or without PSB1115 pretreatment. A cell counting kit-8 (CCK-8) assay was used to evaluate cell proliferation ability, and cell migration ability was determined by cell scratch experiments with HCECs.

Results: Netrin-1 expression decreased slightly after A. fumigatus Infection and then increased to its peak. A2BAR expression increased at 1 day post Infection (p.i.), with a subsequent decline. Compared to the PBS control, exogenous netrin-1 attenuated the inflammatory response, PMN infiltration, and expression of the proinflammatory factors IL-1β and TNF-α, while IL-10 expression was up-regulated. In RAW 264.7 cells, recombinant netrin-1 obviously inhibited the mRNA expression of IL-1β and TNF-α and promoted the mRNA expression of the anti-inflammatory cytokine IL-10. Pretreatment with PSB1115 resulted in disease aggravation and higher levels of the proinflammatory factors IL-1β and TNF-α both in vivo and in vitro. And the effect of netrin-1 on inflammatory factors was abolished by PSB1115. Moreover, compared to the control treatment, exogenous netrin-1 significantly facilitated the proliferation and migration of HCECs.

Conclusions: Netrin-1 attenuates inflammation in C57BL/6 mice infected with A. fumigatus, and it may play this role via the receptor A2BAR. Additionally, netrin-1 can promote the proliferation and migration of HCECs.