Purpose:
CCL2 deficient mice have been shown to have reduced endotoxin–induced uveitis (EIU), and in senescence, to develop cardinal features of age–related macular degeneration (AMD). Variation within CX3CR1 has been associated with human AMD. A new mouse strain, CCL2/CX3CR1 double deficient (DKO), was recently generated by cross–breeding CCL2 deficient (CCL2 KO) and CX3CR1 deficient (CX3CR1 KO) mice, and was shown to develop AMD–like features. This study is to ascertain the ocular immune response in EIU elicited by lipopolysaccharide (LPS) in DKO mice.
Methods:
EIU was induced in a total of 20 DKO, 20 CX3CR1 KO, 18 B6 (CX3CR1 WT), 21 CCL2 KO and 20 CCL2 WT mice following intraperitoneal injection of 100 µg of LPS in 0.1 ml PBS. The control groups (6 for each strain) received PBS alone. All mice were euthanized 24 hours post injection. Eyes were collected for histology examination and RNA isolation for RT–PCR of ß–Actin, IL6, TNFα, IFNγ, CXCL10, CCL5 and TLR4. Real–Time PCR and ΔΔCt methods were used to quantitatively measure the increase of gene expression following LPS injection.
Results:
EIU was significantly reduced in DKO and CX3CR1 KO mice as compared to the other strains. The number of ocular inflammatory cells was 6.7 ± 1.3 (mean ± SE) in DKO, 5.9 ± 1.6 in CX3CR1 KO, 11.3 ± 3.5 in B6, 16.7 ± 2.2 in CCL2 KO and 21.0 ± 4.0 in CCL2 WT mice. The p values were: 0.002, DKO vs CCL2 WT; 0.22, vs B6; 0.162, CX3CR1 KO vs B6 and 0.356, CCL2 KO vs WT. Fold increase of gene transcription in EIU is shown in table1:
Conclusions:
All KO strains showed significant decreases in TLR4, a gene product that regulate LPS response and EIU development. DKO mice had significant hyporeactive ocular response to LPS. Lack of CCL2 and CX3CR1 causes inefficient leukocyte recruitment in response to inflammation and may result in elevated ocular IFNγ and CXCL10, as well as suppressed IL6 and TNFα. CCL5 was only lower in CCL2 KO and DKO mice. The mechanism behind EIU in DKO mice is closely related to TLR4 signaling and monocyte attractants. Investigation into the inflammation observed in this study may reveal some possible mechanisms behind early AMD development.
Keywords: inflammation • uveitis-clinical/animal model • cytokines/chemokines