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. 2013 Oct 4;8(10):e75786.
doi: 10.1371/journal.pone.0075786. eCollection 2013.

Th17 cell-mediated neuroinflammation is involved in neurodegeneration of aβ1-42-induced Alzheimer's disease model rats

Jun Zhang  1 , Kai-Fu Ke, Zhan Liu, Yi-Hua Qiu, Yu-Ping Peng
Affiliations

Th17 cell-mediated neuroinflammation is involved in neurodegeneration of aβ1-42-induced Alzheimer's disease model rats

Jun Zhang et al. PLoS One. .

Abstract

Neuroinflammation, especially innate immunocyte-mediated neuroinflammation, has been reported to participate in pathogenesis of Alzheimer's disease (AD). However, the involvement of adaptive immune cells, such as CD4(+) T lymphocytes, in pathogenesis of AD is not well clarified. Herein, we focus on T helper 17 (Th17) cells, a subpopulation of CD4(+) T cells with high proinflammation, and show the implication of the cells in neurodegeneration of AD. Amyloid β1-42 (Aβ1-42) was bilaterally injected into hippocampus of rats to induce AD. On days 7 and 14 following the Aβ1-42 administration, escape latency of the rats in Morris water maze was increased, expression of amyloid precursor protein was upregulated, but expression of protein phosphatase 2A was downregulated in the hippocampus, and Nissl stain showed neuronal loss and gliosis in CA1 region. Infusion of FITC-linked albumin in blood circulation and combination with immunostaining of hippocampal sections for RORγ, a specific transcriptional factor of Th17 cells, demonstrated blood-brain barrier (BBB) disruption and Th17 cells' infiltration into brain parenchyma of AD rats. Expression of Th17 proinflammatory cytokines, interleukin (IL)-17 and IL-22, was increased in the hippocampus, and concentrations of the two cytokines were elevated in both the cerebrospinal fluid and the serum in AD occurrence and development. Compared with intact or saline-treated control rats, AD animals indicated an upregulated expression of Fas and FasL in the hippocampus. Further, the immunofluorescent histochemistry on AD hippocampal sections with NeuN, RORγ, Fas and FasL displayed that Fas was principally expressed by neurons and FasL was predominantly expressed by Th17 cells, and that neuronal apoptosis shown by TUNEL and NeuN double-labeled cells increased. These results suggest that Th17 cells, which were infiltrated into AD brain parenchyma, participate in neuroinflammation and neurodegeneration of AD by release of proinflammatory cytokines and by direct action on neurons via Fas/FasL apoptotic pathway.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Aβ1-42 treatment of bilateral hippocampus of rats induces AD-like changes.
Rat hippocampus received 1 µl injection containing 4 µg Aβ1-42 on each side. As a control, equal volume of saline was bilaterally injected into the hippocampus. (A) Escape latency in water maze for testing spatial learning and memory of rats. Left panel exhibits swimming tracks of rats in Morris water maze. Within the big rounds, the small rounds are escape platforms. The red dots indicate the starting points of the rats, and the blue dots denote their ending points. The upper row displays the first training tracks of the rats. We see that the rats, regardless of treatments, could almost not find the escape platforms within 60 s. The lower row is the final escape tracks of the rats in the water maze after eight trials, which presents that the rats with the Aβ1-42 treatment need longer time to reach the hidden platforms. The statistics for seven-repeated experiments is shown in right panel. (B) Expression levels of APP, the precursor protein of Aβ, and PP2A, a protein phosphatase that can reduce hyperphosphorylation of tau protein, in the hippocampus. Three or four repetitions were conducted in this experiment. (C) Nessl stain of hippocampal CA1 region of rats. We note that neurons lost and glial cells proliferated in the CA1 region where Aβ1-42 had been injected 7 day or 14 day earlier. The gliosis also occurred around the injection needle tracks, which are indicated by the long arrows. The insets within the images are general views of the hippocampus and the CA1 regions are denoted. *p<0.05, **p<0.01, vs. intact or saline; +p<0.05, vs. AD (day 7). AD (day 7)/AD (day 14): on day 7/on day 14 after Aβ1-42 injection in the hippocampus.
Figure 2
Figure 2. FITC-linked albumin infusion and RORγ immunohistochemistry show BBB disruption and Th17 cell infiltration into brain parenchyma.
FITC-labeled albumin was infused into the carotid artery of rats, and then the hippocampus was cut into 40 µm-thick sections for observation of BBB dysfunction. These sections were additionally stained for RORγ, a specific transcriptional factor of Th17 cells. In the upper panel, the arrows denote the effusion of FITC-linked albumin out of blood vessels, suggesting BBB dysfunction. In the middle panel, the arrows point at RORγ-positive cells, which are more in the hippocampus of AD model rats than in that of intact or saline animals, suggesting that Th17 cells increase in AD brain. In the lower panel, we see by the merged images that the RORγ-positive cells are localized around disrupted BBB, suggesting that the Th17 cells infiltrate into brain parenchyma from the disrupted BBB. This experiment was repeated three times and the same phenomena as shown in the photographs were observed. AD (day 7)/AD (day 14): on day 7/on day 14 after Aβ1-42 injection in the hippocampus.
Figure 3
Figure 3. Elevation of IL-17 and IL-22 levels in the hippocampus, CSF and serum of AD rats.
The hippocampus tissue around needle tract for injection of Aβ1-42 was extracted for measurement of gene and protein expression of Th17 specific cytokines, IL-17 and IL-22. ELISA assay was used to test titers of the two cytokines in the CSF, which was withdrawn by foramen magnum puncture, and in the serum, which was obtained from blood of right ventricle. (A) Gene expression of IL-17 and IL-22 in the hippocampus. (B) Protein expression of the two cytokines in the hippocampus. (C) Concentrations of IL-17 and IL-22 in the CSF and serum. The data are from three- or four-repeated experiments. *p<0.05, **p<0.01, vs. intact or saline. AD (day 7)/AD (day 14): on day 7/on day 14 after Aβ1-42 injection in the hippocampus.
Figure 4
Figure 4. Upregulation of Fas and FasL mRNA and protein expression in the hippocampus of AD rats.
The hippocampus tissue around needle tract for injection of Aβ1-42 was examined for expression of Fas and FasL, the transmembrane molecules known as death receptor and ligand, respectively. An evidently upregulated gene expression (A) and protein expression (B) of the two transmembrane proteins is seen. These effects were larger on day 14 than on day 7 post-Aβ1-42 injection. The data were obtained from three or four replications of the experiments. **p<0.01, vs. intact or saline; ++p<0.01, vs. AD (day 7). AD (day 7)/AD (day 14): on day 7/on day 14 after Aβ1-42 injection in the hippocampus.
Figure 5
Figure 5. Expression and co-localization of Fas and FasL in neurons and Th17 cells in the hippocampus of AD rats.
The hippocampus was coronally cut into 40 µm-thick sections, and these slides were stained with Fas, FasL, NeuN and RORγ by immunofluorescent histochemistry. The immunoreactive cells for Fas, FasL and RORγ are all increased in AD hippocampus, despite the two phases, on days 7 and 14 after Aβ1-42 administration. Fas is more co-localized with NeuN but less co-localized with RORγ. On the contrary, FasL is less co-localized with NeuN but more co-localized with RORγ. The arrows point at the double-stained cells, which are magnified in the insets.
Figure 6
Figure 6. Neuronal apoptosis increases in hippocampal CA1 area of AD rats.
TUNEL staining was conducted following NeuN labeling on the brain sections that mainly displayed hippocampal CA1 area, where Aβ1−42 had been injected 7 days ago. Note that TUNEL-stained cells increase and that some of them are also labeled by NeuN in the CA1 area of AD hippocampus.
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