Knockout of fractalkine receptor Cx3cr1 does not alter disease or microglial activation in prion-infected mice

Abstract

Microglial activation is a hallmark of the neuroimmunological response to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and prion disease. The CX3C chemokine axis consists of fractalkine (CX3CL1) and its receptor (CX3CR1); these are expressed by neurons and microglia respectively, and are known to modulate microglial activation. In prion-infected mice, both Cx3cr1 and Cx3cl1 are altered, suggesting a role in disease. To investigate the influence of CX3C axis signalling on prion disease, we infected Cx3cr1 knockout (Cx3cr1-KO) and control mice with scrapie strains 22L and RML. Deletion of Cx3cr1 had no effect on development of clinical signs or disease incubation period. In addition, comparison of brain tissue from Cx3cr1-KO and control mice revealed no significant differences in cytokine levels, spongiosis, deposition of disease-associated prion protein or microglial activation. Thus, microglial activation during prion infection did not require CX3C axis signalling.

Fig. 1.

Effect of Cx3cr1 knockout on development of scrapie (‘Sc’)-associated clinical signs, weight change and incubation period. (a) Comparison of nesting behaviour scores (5, near perfect nest; 1, no nest). (b) Comparison of percentage weight change in 22L- and RML-infected Cx3cr1-KO mice and C57 mice beginning at preclinical times. Uninfected Cx3cr1-KO mice and C57 mice are shown for comparison. Knockout of Cx3cr1 had no significant effect on weight change. (c) Survival curves showing Cx3cr1-KO versus C57 control mice for 22L and RML scrapie strains. Data are percentage of surviving animals versus days p.i. Mean ± sd incubation period for each strain is set inside each graph. Statistical analysis by Mantel–Cox log-rank analysis indicated no significant differences. (d) Graphs show comparison of five scrapie-associated parameters (a score of 0 was considered normal and a score of 3 was the most advanced stage consistent with a humane point of euthanasia; see Methods for details of scoring). For all graphs, except (c), data are mean ± sd. Numbers of uninfected mice were: C57, n = 4 and Cx3cr1-KO, n = 4. For scrapie-infected mice, respective numbers were (22L, RML): C57, n = 11, 12 and Cx3cr1-KO, n = 10, 12. Statistical differences were evaluated by two-way ANOVA (Prism software; GraphPad).

Fig. 2.

PrP^Sc immunoblot and neuropathology in 22L-infected mice. (a) Immunoblots of brain homogenates from clinical 22L-infected C57 mice (n = 3) and Cx3cr1-KO mice (n = 3) and one uninfected C57 mouse. Equivalent loads of brain homogenate were treated for 1 h with 50 mg proteinase K ml^− 1, run on SDS-PAGE gels, blotted and probed with anti-PrP antibody D13, and exposed with chemiluminescent reagents. Lanes 1, 2 and 3 are similar to lanes 5, 6 and 7, suggesting equivalent amounts of PrP^Sc deposition in Cx3cr1-KO and C57 mice. (b) Histopathological analysis of brain from clinical 22L-infected C57 and Cx3cr1-KO mice. An uninfected Cx3cr1-KO age-matched mouse is shown as a control. These images are representative of six C57 mice and five Cx3cr1-KO mice analysed. The top row shows comparative staining of whole-brain sagittal sections for PrP^Sc using anti-PrP antibody D13 (brown). The inset images show close-up views of PrP^Sc deposition in the thalamus. The second row shows vacuolation in the thalamus using haematoxylin and eosin (H&E) staining. The third row compares brain sagittal sections stained with anti-IBA1 antibody (red), which highlights microglial morphology and distribution. Inset images show close-ups of the anti-IBA1-positive microglia in the thalamus. Note the large cell bodies with short, thickened processes, indicative of microglial activation, in the infected mice. In contrast, microglia in the uninfected mouse have small cell bodies with long, thin, highly ramified processes. The bottom row compares astrogliosis in brain sagittal sections by staining with anti-GFAP antibody (red). Images are for 22L only; analysis of RML-infected brain gave similar results (data not shown).

Fig. 3.

Elevated cytokine levels in the brain of scrapie-infected mice at clinical time point. Using multiplex immunoassays, protein levels of 23 cytokines [eotaxin, granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17A, KC, CCL2, CCL3, CCL4, CCL5 and TNF-α] were measured in brain homogenates from scrapie-infected mice taken at the clinical end-point. Levels of 10 cytokines that were elevated in 22L and RML-infected C57 and Cx3cr1-KO mice compared with mock are shown here. Levels of cytokines in scrapie-infected Cx3cr1-KO mice were not significantly different from those of scrapie-infected C57 mice, except for CCL2 levels for scrapie strain 22L. Each dot represents one mouse and data are presented as mean ± sd. Statistical differences were evaluated by one-way ANOVA (Prism software; GraphPad).