An enhanced inflammatory response in the aged choroid is associated with the upregulation of CCL2 and neutrophil markers following the induction of choroidal neovascularisation

Abstract

Purpose: CNV severity increases with age but the kinetics and mechanism for this are not fully understood. We hypothesised that age-related chronic inflammatory changes in the chorioretinal complex of the eye drive the recruitment of myeloid cells that modulate CNV severity.

Methods: We determined the kinetics of CNV growth in young (<4 months) and old (>18 months) wild-type mice using serial fundus fluorescein angiography. Based on the results we induced choroidal neovascularisation (CNV) at 10 locations in the fundi of young and old wild-type animals and harvested retinal pigment epithelium/choroid for analysis by quantitative reverse-transcriptase polymerase chain reaction of VEGF, CD11b, CCL2, CCR2, CXCR1, CXCR2, CX3CR1, TNFa, IFNg, Il-4, Il-6, Il-8 and Il-10.

Results: We found the myeloid cell chemokine CCL2 to be undetectable in young choroid but highly upregulated in tissue derived from aged mice. We also found the myeloid cell marker CD11b and the chemokine receptors CXCR1, CXCR2 and CX3CR1 to be upregulated in the aged unlasered choroid, indicating that myeloid cell numbers in this tissue increase with age. Analysis of choroid at 3 days post-induction of CNV revealed 40-fold higher levels of expression of CCL2 in aged compared with young animals. This was associated with a further increase in CXCR2 together with elevated CCR2 in lasered, aged tissue. CXCR2 is highly expressed on neutrophils while CCR2 is expressed on both ‘inflammatory’ monocytes and neutrophils entering an inflamed microenvironment. These results therefore suggest an enhanced recruitment of both CCR2+ myeloid cells populations to sites of CNV in aged animals. We also observed higher levels of expression of the pro-inflammatory cytokine TNFa and the pro-angiogenic cytokines Il-6 and Il-10 in aged choroid compared with young at 3 days post-induction of CNV.

Conclusions: Levels of CCL2 in the aged choroid are elevated and the tissue is therefore primed for myeloid cell recruitment. The induction of CNV in aged eyes results in further upregulation of CCL2 and its cognate receptor CCR2 at 3 days post laser. The strong upregulation of chemokine receptors (CXCR2 and CXCR1) most highly expressed on neutrophils suggests that these cells may contribute to the increased inflammatory response, and therefore CNV severity, in aged animals.

Keywords: 412 age-related macular degeneration • 413 aging • 453 choroid: neovascularization