Toll-like receptor 4-dependent upregulation of cytokines in a transgenic mouse model of Alzheimer's disease

1742-2094-5-23 1742-2094 Research Toll-like receptor 4-dependent upregulation of cytokines in a transgenic mouse model of Alzheimer's disease Jin Jing-Ji jingji@uic.edu Kim Hong-Duck hongduckkim@gmail.com Maxwell J Adam jadammaxwell@gmail.com Li Ling lili@uab.edu Fukuchi Ken-ichiro kfukuchi@uic.edu

Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Box 1649, Peoria, IL 61656, USA

Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-0024, USA

Journal of Neuroinflammation 1742-2094 2008 5 1 23 http://www.jneuroinflammation.com/content/5/1/23 18510752 10.1186/1742-2094-5-23 13 2 2008 29 5 2008 29 5 2008 2008 Jin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Aβ deposits in the brains of patients with Alzheimer's disease (AD) are closely associated with innate immune responses such as activated microglia and increased cytokines. Accumulating evidence supports the hypothesis that innate immune/inflammatory responses play a pivotal role in the pathogenesis of AD: either beneficial or harmful effects on the AD progression. The molecular mechanisms by which the innate immune system modulates the AD progression are not well understood. Toll-like receptors (TLRs) are first-line molecules for initiating the innate immune responses. When activated through TLR signaling, microglia respond to pathogens and damaged host cells by secreting chemokines and cytokines and express the co-stimulatory molecules needed for protective immune responses to pathogens and efficient clearance of damaged tissues. We previously demonstrated that an AD mouse model homozygous for a destructive mutation of TLR4 has increases in diffuse and fibrillar Aβ deposits as well as buffer-soluble and insoluble Aβ in the brain as compared with a TLR4 wild-type AD mouse model. Here, we investigated the roles of TLR4 in Aβ-induced upregulation of cytokines and chemokines, Aβ-induced activation of microglia and astrocytes and Aβ-induced immigration of leukocytes.

Methods

Using the same model, levels of cytokines and chemokines in the brain were determined by multiplex cytokine/chemokine array. Activation of microglia and astrocytes and immigration of leukocytes were determined by immunoblotting and immunohistochemistry followed by densitometry and morphometry, respectively.

Results

Levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-10 and IL-17 in the brains of TLR4 wild-type AD mice were significantly higher than those in TLR4 wild-type non-transgenic littermates. Such increases in cytokines were not found in TLR4 mutant AD mice as compared with TLR4 mutant non-transgenic littermates. Although expression levels of CD11b (a microglia marker) and GFAP (a reactive astrocyte marker) in the brains of TLR4 mutant AD mice were higher than those in TLR4 wild type AD mice, no difference was found in levels of CD45 (common leukocyte antigen).

Conclusion

This is the first demonstration of TLR4-dependent upregulation of cytokines in an AD mouse model. Our results suggest that TLR4 signaling is involved in AD progression and that TLR4 signaling can be a new therapeutic target for AD.

Background

Patients with Alzheimer's disease (AD) develop deposits of aggregated amyloid β-protein (Aβ) in neuritic plaques and cerebral vessels (cerebrovascular amyloid angiopathy). Fibrillar Aβ deposits in AD brain are accompanied by innate immune responses such as activated microglia and increased levels of cytokines 1. Earlier studies indicated that the deposition of Aβ in the brain might activate microglia, initiating a proinflammatory cascade that resulted in the release of potentially cytotoxic molecules, cytokines, complements, proteases and other acute phase proteins, ultimately causing neurodegeneration 12. In accordance with this view, long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduced the risk of AD and delayed its onset 345678. Overexpression of mutant forms of amyloid β-protein precursor (APP) in the brains of transgenic mice produced amyloid plaques surrounded by activated microglia and reactive astrocytes and upregulated interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α, which resembled the alterations found in patients with AD 9. When Tg2576 mice, an AD mouse model, overexpressing a mutant form of APP were treated with an NSAID, ibuprofen, the mice improved in open field activity and had reductions in levels of IL-1β, reactive astrocytes and Aβ load 1011. The deletion of the TNF type 1 death receptor gene in APP23 transgenic mice, another transgenic mouse model of AD, inhibited Aβ generation, decreased amyloid plaques and prevented learning and memory deficits 12. The deletion of the interferon (IFN)-γ receptor type I gene in Tg2576 mice caused less gliosis and amyloid plaques. In IFN-γ receptor wild-type Tg2576 mice, IFN-γ elicited TNF-α secretion resulting in upregulation of β-site APP-cleaving enzyme (BACE1) in astrocytes, which caused an increase in Aβ production. Additionally, upregulation of IFN-γ and TNF-α suppressed Aβ degradation by microglia in the latter mice 13. These observations support the notion that upregulation of proinflammatory cytokines and activation of glial cells promote the disease progression.

Recently, however, the reports of the potential beneficial effects of innate immune responses in AD are increasing. Activation of cultured microglia with toll-like receptor 2 (TLR2), TLR4, or TLR9 ligand markedly boosted ingestion of Aβ 14151617. An acute injection of lipopolysaccharide (LPS, a TLR4 ligand) in the hippocampus reduced Aβ load in an AD transgenic model 18 and microglial activation is required for the LPS-induced reduction of Aβ load 19. Accumulation of Aβ in the brain of an AD mouse model triggered chemoattraction of bone marrow-derived cells (microglia) that restricted amyloid deposits in the brain 20. Upon activation with LPS, bone marrow-derived microglia decreased Aβ load in the brain of an AD mouse model 21. Mononuclear cells from normal subjects were able to clear Aβ from the sections of AD brain but those from AD patients were not. Furthermore, Aβ upregulates expression of β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (MGAT3) and TLRs in mononuclear cells from normal subjects, whereas these genes were down-regulated in cells from AD patients 22, suggesting immune defects in AD patients. The deletion of the CCR2 [a receptor for monocyte chemoattractant protein-1 (MCP-1 or CCL2)] gene in Tg2576 mice reduced the number of microglia and impaired Aβ clearance in the brain 23. Overexpression of C3 inhibitory soluble complement receptor-related protein inhibited C3 in the brain of an AD mouse model, decreased microglial activation and increased Aβ deposition and neurodegeneration 24. Overproduction of transforming growth factor (TGF)-β1 in an AD mouse model resulted in a vigorous microglial activation that was accompanied by at least a 50% reduction in Aβ load 25. Overexpression of IL-1β in the hippocampus of APPswe PS1dE9 mouse model of AD 26 activated microglia and astrocytes, upregulated expression of TNF-β and IL-6 and decreased amyloid plaques 27. These reports suggest that neuroinflammation found in AD brain is beneficial and that activation of microglia and astrocytes as well as upregulation of cytokines and chemokines can be therapeutic.

TLRs function as pattern-recognition receptors in the innate immune system 28. Activated phagocytes and tissue dendritic cells through TLR signaling respond to pathogens and damaged host cells by secreting chemokines and cytokines and express the co-stimulatory molecules needed for protective immune responses, efficient clearance of damaged tissues and adaptive immunity 293031. Microglia is the phagocyte in the central nervous system and activated by a number of TLR ligands 3233. We previously demonstrated that an AD mouse model (Mo/Hu APPswe PS1dE9 mice) homozygous for a destructive mutation of TLR4 (Tlr^Lps-d/Tlr^Lps-d) had increases in diffuse and fibrillar Aβ deposits as well as buffer-soluble and insoluble Aβ in the brain as compared with a TLR4 wild-type AD model mice 17. Because induction of TLR signaling activates microglia and upregulates cytokines and chemokines, in the present study, we investigated the roles of TLR4 in activation of microglia and astrocytes as well as upregulation of cytokines and chemokines in the AD mouse model. Activation of microglia and astrocytes was determined by immunohistochemistry and immunoblotting. Levels of cytokines and chemokines were quantified by multi-plex cytokine array.

Methods

Animals

A pathogen-free transgenic line of AD mouse model, Mo/Hu APPswe PS1dE9 mice 26, was obtained from Jackson Laboratory (Bar Harbor, ME) and maintained by crossing transgenic males with B6C3F1 females that were purchased from Jackson Lab, also. C3H/HeJ mice are highly susceptible to Gram-negative infection and resistant to bacterial lipopolysaccharide (LPS) due to a destructive mutation of the TLR4 gene (TlrLps-d). The genotyping for the APPswe transgene was performed by the PCR-based method provided by the Jackson Lab. The TLR4 genotype was determined by PCR followed by restriction enzyme digestion with Nla III as described previously 17. The transgenic mice express chimeric mouse/human APP with the double mutations (K670N and M671L) and human PS1 with a deletion of exon 9 found in familial AD patients. In this study, four experimental groups of 13–15 month-old mice which differed in the TLR4 gene and Mo/Hu APPswe PS1dE9 transgene were used. The four experimental groups were (1) homozygous TLR4 mutant Mo/Hu APPswe PS1dE9 transgenic mice (n = 8), designated here as TLR4m AD mice for simplicity, (2) TLR4 wild-type transgenic mice (n = 10), designated as TLR4w AD mice, (3) homozygous TLR4 mutant non-transgenic littermates (n = 9), designated as TLR4m non-AD mice and (4) TLR4 wild-type non-transgenic mice (n = 9), designated as TLR4w non-AD mice.

Brain tissue lysate preparation

Mice were sacrificed by lethal injection of pentobarbital and the brains were quickly removed. After removing the cerebellums, the left hemispheres were processed for protein array and immunoblot analyses. The left cerebrum was lysed using the Bio-Plex cell lysis kit (Bio-Rad, Hercules, CA). Briefly, the cerebrum was rinsed with the cell wash buffer and cut into 3 × 3 mm pieces. The sample was transferred to a Dounce homogenizer on ice and homogenized in the lysis buffer containing proteinase inhibiters. Then the ground tissue sample was transferred to a clean microcentrifuge tube and frozen at -80°C. After thawing, the sample was sonicated on ice for 30 seconds. The sample was centrifuged at 4,500 g for 10 minutes at 4°C, and the supernatant was collected for the protein array and immunoblotting.

Immunoblotting and densitometric analysis

Immunoblot analysis was used to quantify cerebral CD11b, CD45 and glial fibrillary acidic protein (GFAP) in AD mice. Protein concentrations of brain lysates were determined by Bio-Rad Protein Assay (Bio-Rad). Five micrograms of total protein from each sample were applied to 10–20% Tris-HCl gradient SDS-PAGE and electrotransferred to polyvinylidine difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked by phosphate buffered saline (PBS) containing 5% non-fat dried milk (w/v), 0.02% sodium azide, and 0.02% Tween 20 for 1 hr at room temperature, incubated at 4°C overnight with primary antibodies CD11b (Serotec, MCA711, Raleigh, NC) for detection of activated microglia, CD45 (Serotec, MCA1031G) for detection of migratory leukocytes or GFAP (Dako, Demark) for reactive astrocytes and visualized by the western lighting chemiluminescence reagent plus (Perkin Elmer, Boston, MA) according to the manufacturer's protocol. The membranes were reprobed with monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon, Temecula, CA). The optical densities of CD11b, CD45, GFAP and GAPDH bands from the membranes were determined by densitometric scanning using a HP Scanjet G3010 Photo Scanner and HP Photosmart Software. The optical density of each protein band was divided by that of the GAPDH band on the same lane from the same membrane for normalization. The ratios were expressed as mean ± standard error. Intergroup differences were assessed by analysis of variance (ANOVA) and two-tailed Student's t-test. P < 0.05 was considered statistically significant.

Cytokine/chemokine microarray

Cerebral tissue lysates were prepared and their protein concentrations were determined as described above. Levels of cytokines and chemokines in the cerebral tissue lysates were determined by the bead-based suspension microarray technology (AssayGate, Ijamsville, MD) 34. The threshold of detection was determined by adding two standard deviations to the mean fluorescence intensity of twenty zero standard (background) replicates. The minimum detectable concentrations were 3.6 pg/ml for IL-1α, 3.8 pg/ml for IL-1β, 2.3 pg/ml for IL-2, 2.5 pg/ml for IL-3, 4.3 pg/ml for IL-4, 1.3 pg/ml for IL-6, 8.2 pg/ml for IL-10, 11 pg/ml for IL-12 (p40), 3.5 pg/ml for IL-12(p70), 9.2 pg/ml for IL-17, 6.6 pg/ml for granulocyte macrophage colony-stimulating factor (GM-CSF), 2.3 pg/ml for IFN-γ, 16 pg/ml for TGF-β1, 3.7 pg/ml for TNF-α, 2.5 pg/ml for MCP-1, 1.3 pg/ml for macrophage inflammatory protein (MIP)-1α and 3.1 pg/ml MIP-1β. The results were expressed in picograms per milligram of brain protein and analyzed by ANOVA and two-tailed Student's t-test using SigmaStat for Windows Version 3.00. P < 0.05 was considered statistically significant.

Immunohistochemistry

Frozen sections (5 μm thickness) were prepared and subjected to the immunoperoxidase method. Endogenous peroxidase was eliminated by treatment with 3% H₂O₂/10% methanol Tris-buffered saline (TBS) for 20 min at room temperature. After washing with water and 0.1 M TBS (pH 7.4), slides were blocked with 2% bovine serum albumin (BSA) and 2% goat serum in 0.1% triton-X-100 TBS (TBST) buffer for 60 min at room temperature to prevent non specific protein binding. Then the slides were incubated with primary antibody CD11b (1:200), CD45 (1: 1000), or GFAP (1:1000) in 2% BSA, 2% goat serum TBST overnight at 4°C. The sections were rinsed in 0.1 M TBST containing 0.1% BSA and incubated with biotinylated secondary antibody anti-rat IgG (1:200) for CD11b, anti-rat IgG (1:1500) for CD45 or anti-rabbit IgG (1:1000) for GFAP in 2% BSA, 1% goat serum TBST for 1 h at room temperature. Finally, the avidin biotin peroxidase method using 3,3'-diaminobenzidine as a substrate (Vector, Burlingame, CA) was performed according to manufacturer's protocol. For the negative control, slides were processed without primary antibody.

Histomorphometry for quantification of activated microglia and reactive astrocytes was performed using an OLYMPUS IX71 microscope, OLYMPUS DP70 digital camera and the Image Pro Plus v4 image analysis software (Media Cybernetics, Silver Spring, MD) capable of color segmentation and automation via programmable macros. Four to five coronal brain sections, each separated by more than 240 μm interval, from each mouse were analyzed. All brain sections contained both cerebral cortex and hippocampus. Consecutive 4 pictures of the cerebral cortex with no overlaps, starting from the midline were taken from each brain section using a 10× objective and 1× eyepiece lens. Pictures in which folded tissues were found were discarded. Thus, approximately 15 pictures of the cerebral cortex from each mouse were analyzed. The area of the cerebral cortex in the pictures varied from approximately 0.5 mm²to 1 mm². CD11b, CD45 and GFAP stained areas were expressed as a percentage of total cerebral cortex examined. Data were expressed as mean ± standard error as a bar graph. Intergroup differences were assessed by ANOVA and two-tailed Student's t-test. P < 0.05 was considered statistically significant.

Results

Activated microglia

Activated microglia were analyzed for expression of CD11b (Mac-1) by immunohistochemistry. Representative micrographs from the cerebral cortex of AD mice were shown in Fig. 1 A through D. Morphometric analysis of the CD11b immunostaining revealed that the cerebral cortex of TLR4m AD mice (1.10 ± 0.18%) had greater microglial immunoreactivity than TLR4w AD mice (0.45 ± 0.10%, P = 0.021) (Fig. 1E). CD11b immunoreactivity in the brains of TLR4m and TLR4w non-AD mice was unremarkable (data not shown). Microglial activation was further determined by immunoblot analysis (Fig. 2). Expression levels of CD11b in the cerebral tissue lysates from TLR4m AD mice were on average higher than those from TLR4w AD mice, which were not statistically significant due to large variances (Fig. 2).

Figure 1

CD11b immunoreactive microglia in TLR4m and TLR4w AD mice

CD11b immunoreactive microglia in TLR4m and TLR4w AD mice. The frozen sections of cerebral cortices from TLR4m (A and B) and TLR4w (C and D) AD mice were stained with anti-CD11b antibody. Scale bars are 50 μm in A and C, and 10 μm in B and D. The middle images (B and D) are a high magnification of the areas indicated by the squares in the top images (A and C). CD11b immunoreactive area percentages of TLR4m and TLR4w AD mice were shown as a bar graph (means ± SE, * P < 0.05) (E). Approximately 15 fields of the cerebral cortex (0.5 to 1 mm²each, using a 10× objective and 1× eyepiece lens) from 4 – 5 coronal brain sections, each separated by more than 240 μm interval, from each mouse were analyzed. TLR4m and TLR4w represent TLR4m and TLR4w AD mice, respectively.

Figure 2

Levels of CD11b and GAPDH in the cerebral tissue lysates were determined by immunoblotting using anti-CD11b and anti-GAPDH antibodies, respectively

Levels of CD11b and GAPDH in the cerebral tissue lysates were determined by immunoblotting using anti-CD11b and anti-GAPDH antibodies, respectively. The bar graph represents densitometric quantification of CD11b after normalization with GAPDH (means ± SE). The mean of CD11b levels in TLR4m AD mice is greater than that in TLR4w AD mice but not statistically significant. Lane 1 through 4 are tissue lysates from TLR4m AD mice and lane 5 through 9 are from TLR4w AD mice.

Migratory leukocytes

The common leukocyte antigen, CD45, was used to assess the invasion of the brain by blood-derived leukocytes. The cerebral cortex of TLR4m and TLR4w AD mice was immunostained with anti-CD45 antibody (Fig. 3 A through D). No significant difference was observed between the two groups: 3.74 ± 0.19 and 3.54 ± 0.12% for TLR4m and TLR4w AD mice, respectively (P = 0.37) (Fig. 3E). CD45 immunoreactivity in the brains of TLR4m and TLR4w non-AD mice was unremarkable (data not shown). The common leukocyte antigen in the cerebral tissue lysates was further determined by immunoblot analysis (Fig. 4). There was no difference between TLR4m and TLR4w AD mice in expression levels of CD45 (Fig. 4).

Figure 3

CD45-immunoreactive cells in TLR4m and TLR4w AD mice

CD45-immunoreactive cells in TLR4m and TLR4w AD mice. The frozen sections of cerebral cortices from TLR4m (A and B) and TLR4w (C and D) AD mice were stained with anti-CD45 antibody. Scale bars are 50 μm in A and C, and 10 μm in B and D. The middle images (B and D) are a high magnification of the areas indicated by the squares in the top images (A and C). CD45 immunoreactive area percentages of TLR4m and TLR4w AD mice were shown as a bar graph (means ± SE) (E). Approximately 15 fields of the cerebral cortex (0.5 to 1 mm²each, using a 10× objective and 1× eyepiece lens) from 4 – 5 coronal brain sections, each separated by more than 240 μm interval, from each mouse were analyzed. TLR4m and TLR4w represent TLR4m and TLR4w AD mice, respectively.

Figure 4

Levels of CD45 and GAPDH in the cerebral tissue lysates were determined by immunoblotting using anti-CD45 and anti-GAPDH antibodies, respectively

Levels of CD45 and GAPDH in the cerebral tissue lysates were determined by immunoblotting using anti-CD45 and anti-GAPDH antibodies, respectively. The bar graph represents densitometric quantification of CD45 after normalization with GAPDH (means ± SE). The mean of CD45 levels in TLR4m AD mice is greater than that in TLR4w AD mice but not statistically significant. Lane 1 through 4 are tissue lysates from TLR4m AD mice and lane 5 through 9 are from TLR4w AD mice.

Reactive astrocytes

Reactive astrocytes in the brains of TLR4m and TLR4w AD mice were detected by immunohistochemistry using an antibody against an astrocyte marker, GFAP (Fig. 5A through 5D). Morphometric analysis indicated that the cerebral cortex of TLR4m AD mice (1.20 ± 0.07%) had a greater degree of astrocytosis compared with TLR4w AD mice (0.61 ± 0.08%, P = 0.0006) (Fig. 5E). Astrocyte immunoreactivity for GFAP in the cortex of non-AD mice was unremarkable (data not shown). Immunoblot analysis was also used to determine GFAP expression levels (Fig. 6). Densitometric scanning of the immunoblot confirmed that GFAP expression levels in the cerebral tissue lysates from TLR4m AD mice were higher than those from TLR4w AD mice (P = 0.016) (Fig. 6). Thus, astrocytosis in TLR4m AD mice was greater than that in TLR4w AD mice. These results are in agreement with our previous report that Aβ load in the cerebral cortex of TLR4m AD mice is greater than that of TLR4w AD mice 17.

Figure 5

Reactive astrocytes in TLR4m AD mice increase compared to TLR4w AD mice by immunohistochemistry

Reactive astrocytes in TLR4m AD mice increase compared to TLR4w AD mice by immunohistochemistry. The frozen sections of cerebral cortices from TLR4m (A and B) and TLR4w (C and D) AD mice were stained with anti-GFAP antibody. Scale bars are 50 μm in A and C, and 10 μm in B and D. The middle images (B and D) are a high magnification of the areas indicated by the squares in the top images (A and C). GFAP immunoreactive area percentages of TLR4m and TLR4w AD mice were shown as a bar graph (means ± SE, **P < 0.001) (E). Approximately 15 fields of the cerebral cortex (0.5 to 1 mm²each, using a 10× objective and 1× eyepiece lens) from 4 – 5 coronal brain sections, each separated by more than 240 μm interval, from each mouse were analyzed. TLR4m and TLR4w represent TLR4m and TLR4w AD mice, respectively.

Figure 6

Levels of GFAP and GAPDH in the cerebral tissue lysates were determined by immunoblotting using anti-GFAP and anti-GAPDH antibodies, respectively

Levels of GFAP and GAPDH in the cerebral tissue lysates were determined by immunoblotting using anti-GFAP and anti-GAPDH antibodies, respectively. The bar graph represents densitometric quantification of GFAP after normalization with GAPDH (means ± SE). GFAP levels in TLR4m AD mice are greater than those in TLR4w AD mice (*P < 0.05). Lane 1 through 4 are tissue lysates from TLR4m AD mice and lane 5 through 7 are from TLR4w AD mice.

Levels of cytokines and chemokines

Levels of cytokines and chemokines in the cerebral extracts from the four experimental groups of mice were determined by multiplex cytokine/chemokine array analysis using the bead-based array technology 34. Cytokines included IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12 (p40), IL-12(p70), IL-17, GM-CSF, IFN-γ, TGF-β1, and TNF-α. Chemokines determined were MCP-1, MIP-1α and MIP-1β. IL-3 and IL-4 in the cerebral tissue lysates were less than 2.5 and 4.3 pg/ml, respectively, which are the minimum detectable levels. Therefore, IL-3 and IL-4 were omitted from analysis. The levels of these cytokines and chemokines are presented as bar graphs in Fig. 7 and 8. Levels of IL-1β, IL-10, IL-17 and TNF-α increased in the cerebrum of TLR4w AD mice compared to TLR4w non-AD mice (P = 0.029, 0.013, 0.031 and 0.001, respectively) (Fig. 7). There were no significant differences in the other cytokines and chemokines between these TLR4w groups. Levels of MIP-1α increased in TLR4m AD mice compared to TLR4m non-AD mice (P = 0.006) (Fig. 8). There were no significant differences in any of the other cytokines and chemokines between these TLR4m groups. Levels of TNF-α and MIP-1β in TLR4w AD mice were higher than those in TLR4m AD mice (P = 0.014 and 0.019, respectively). There were no significant differences in the other cytokines and chemokines between these transgenic groups. Although levels of many cytokines such as IL-1, IL-2, IL-6, IL-10, IL-12, IL-17 and TGF-β1 in TLR4m non-AD mice on average are higher than those in TLR4w non-AD mice, the differences are not statistically significant. Levels of TNF-α in TLR4m non-AD mice, however, were higher than those in TLR4w non-AD mice (P = 0.048), suggesting compensatory upregulation of some cytokines in TLR4m mice.

Figure 7

Levels of cerebral cytokines and chemokines in TLR4w AD and non-AD mice at the age of 13–15 months

Levels of cerebral cytokines and chemokines in TLR4w AD and non-AD mice at the age of 13–15 months. Levels of cytokines and chemokines in the cerebral tissue lysates were determined by multiplex cytokine/chemokine array analysis. Hatched and open bars represent levels of cytokines and chemokines in TLR4w AD and non-AD mice, respectively. Mean concentrations ± SE are expressed in picograms per milligram of brain protein. *P < 0.05, **P = 0.001.

Figure 8

Levels of cerebral cytokines and chemokines in TLR4m AD and non-AD mice at the age of 13–15 months

Levels of cerebral cytokines and chemokines in TLR4m AD and non-AD mice at the age of 13–15 months. Levels of cytokines and chemokines in the cerebral tissue lysates were determined by multiplex cytokine/chemokine array analysis. Hatched and open bars represent levels of cytokines and chemokines in TLR4m AD and non-AD mice, respectively. Mean concentrations ± SE are expressed in picograms per milligram of brain protein. *P < 0.05.

Discussion

Neuroinflammation characterized by increases in activated microglia and cytokines is thought to modulate the AD progression 12353637. Such neuroinflammatory changes in AD are partly recapitulated in transgenic animal models of AD 9. Here we demonstrated that levels of IL-1β, IL-10, IL-17 and TNF-α increased in the cerebrum of TLR4w AD mice compared to TLR4w non-AD mice. The levels of such cytokines in TLR4m AD mice were not different from those in TLR4m non-AD mice. These results indicate that TLR4 signaling mediates Aβ-induced upregulation of IL-1β, IL-10, IL-17 and TNF-α in TLR4w AD mice. It has been shown that fibrillar Aβ interacted with CD14 (TLR2 and TLR4 accessory protein) and was phagocytosed by cultured microglia and monocytes, resulting in activation of the cells as well as increased secretion of cytokines 3839. Aggregated Aβ failed to activate cultured microglia from TLR4m C3H/HeJ mice resulting in a diminished release of IL-6, TNF-α and nitric oxide compared to microglia from TLR4w C3H/HeN mice 40. TLR4 neutralizing antibody also blocked Aβ-induced activation of human monocytes 40. Upregulation of TLR4 and the association of CD14 with microglia in the brains of AD patients were demonstrated by immunohistochemistry 3840. We also found TLR4 upregulation in activated microglia which were intimately associated with Aβ deposits in Tg2576 mice (data not shown). These findings suggest that aggregated Aβ can induce activation of microglia and upregulation of certain cytokines through TLR4 signaling.

IL-1β, IL-10, IL-17 and TNF-α were upregulated in the brains of AD mice in a TLR4-dependent manner. As demonstrated by many investigators, expression of IL-1β and TNF-α is induced in cultured microglia and astrocytes in response to Aβ treatment 237. Many investigators reported upregulation of IL-1β and TNF-α in AD mouse models and AD patients although some reports presented controversial data 2937. Upregulation of IL-10 was also found in tissue samples from AD mouse models and AD patients 414243. Because cytokines regulate the intensity and duration of immune responses 44 and because overexpression of IL-1β and TGF-β reduced Aβ load in the brains of AD mouse models, upregulation of some of these cytokines might have contributed to reducing Aβ load in TLR4w AD mice 17.

To the best of our knowledge, upregulation of IL-17 has not been reported in tissue samples from AD mouse models and AD patients. IL-17 is a proinflammatory cytokine produced from Th17 T cells that develop from a lineage distinct from the T helper-type 1 and 2 lineages 45. IL-17 has been demonstrated to play important roles in the pathogenesis of autoimmune diseases such as multiple sclerosis and its animal models, experimental autoimmune encephalomyelitis 464748. Upon stimulation with IL-1β, IL-17 is produced by microglia, also 49. Therefore, upregulation of IL-17 may be due to increased levels of IL-1β in the brains of TLR4w AD mice. The role of IL-17 in the pathogenesis of AD remains to be investigated.

Activated microglia and reactive astrocytes are closely associated with fibrilar Aβ deposits but are rarely found in immediate proximity to diffuse Aβ deposits 9. TLR4m AD mice at 14–16 months of age showed an increase in fibrillar Aβ deposits compared to TLR4w mice 17. Immunoreactivity for CD11b and GFAP, markers for activated microglia and astrocyte, respectively, increased in 13–15 month-old TLR4m AD mice compared to TLR4w AD mice. On the other hand, immunoreactivity for CD45 (migratory leukocytes) in TLR4m AD mice was not different from that in TLR4w AD mice. In CCR2-deficient or C3 inhibitor-overexpressing mice, however, an increase in Aβ levels in the brain was associated with a decrease in microglial cells 2324. Overexpression of TGF-β1 or IL-1β increased activated microglia and reduced Aβ load in the brains of AD mouse models 2427. Therefore, we expected less activated microglia and more Aβ load in TLR4m AD mice compared to TLR4w AD mice. The reasons for this discrepancy are not clear. This discrepancy, however, may be due to use of CD11b as a marker for activated microglia because microglia at distinct activation states differentially express several activation markers 919. Alternatively, an acute state of activated microglia needs to be investigated to observe less activated microglia in TLR4m mice because AD mice develop amyloid deposits as early as 4–5 months of age 2650. Different aggregation states of Aβ react with multiple cell surface proteins/receptors other than CD14/TLR4 51. Such proteins/receptors may activate microglia in a chronic state without the beneficial effects. We are currently investigating activation of microglia and expression of cytokines in TLR4m AD mice at younger ages.

Conclusion

In summary, we found upregulation of IL-1β, IL-10, IL-17 and TNF-α in TLR4w AD mice compared to non-AD mice. Such upregulation was not observed in TLR4m AD mice. Our results suggest that TLR4 signaling may be involved in the AD progression through upregulation of certain cytokines. Because IL-1β and TNF-α can modulate the AD-like pathology progression in AD mouse models, TLR4 signaling can be a new therapeutic target for AD.

Abbreviations

AD: Alzheimer's disease; TLRs: toll-like receptors; TNF: tumor necrosis factor; IL: interleukin; Aβ: aggregated amyloid β-protein; NSAID: nonsteroidal anti-inflammatory drugs; APP: amyloid β-protein precursor; IFN: interferon; BACE1: β-site APP-cleaving enzyme; TLR2: toll-like receptor 2; LPS: lipopolysaccharide; MGAT3: mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase; GFAP: glial fibrillary acidic protein; PVDF: polyvinylidine difluoride; PBS: phosphate buffered saline; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ANOVA: analysis of variance; TBS: Tris-buffered saline; BSA: bovine serum albumin; TBST: triton-X-100 TBS; GM-CSF: granulocyte macrophage colony-stimulating factor; TGF: transforming growth factor; MCP-1: monocyte chemotactic protein-1; MIP: macrophage inflammatory protein

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

KF designed the study and reviewed the data, KF and JJ wrote the manuscript, JJ and HDK performed immunohistochemistry and protein assays, JJ performed western blotting, morphometric and statistical analyses, JAM performed production and genotyping of experimental animals, LL provided experimental animals and consultation and reviewed the data.

Acknowledgements

We thank Drs. David Borchelt and Joanna Jankowsky for providing the Mo/Hu APPsw PS1dE9 mice and Linda Walter for her help with preparing this manuscript. This research is supported in part by NIH AG029818 and EY018478, and Alzheimer's Association IIRG-07-59494 and NIRG-06-27725.

Inflammation and Alzheimer's disease Akiyama H Barger S Barnum S Bradt B Bauer J Cole GM Cooper NR Eikelenboom P Emmerling M Fiebich BL Finch CE Frautschy S Griffin WS Hampel H Hull M Landreth G Lue L Mrak R Mackenzie IR McGeer PL O'Banion MK Pachter J Pasinetti G Plata-Salaman C Rogers J Rydel R Shen Y Streit W Strohmeyer R Tooyoma I Van Muiswinkel FL Veerhuis R Walker D Webster S Wegrzyniak B Wenk G Wyss-Coray T Neurobiol Aging 2000 21 383 421 10.1016/S0197-4580(00)00124-X 10858586 Inflammatory processes in Alzheimer's disease Heneka MT O'Banion MK J Neuroimmunol 2007 184 69 91 10.1016/j.jneuroim.2006.11.017 17222916 Delayed onset of Alzheimer's disease with nonsteroidal anti-inflammatory and histamine H2 blocking drugs Breitner JC Welsh KA Helms MJ Gaskell PC Gau BA Roses AD Pericak-Vance MA Saunders AM Neurobiol Aging 1995 16 523 530 10.1016/0197-4580(95)00049-K 8544901 Inverse association of anti-inflammatory treatments and Alzheimer's disease: initial results of a co-twin control study Breitner JC Gau BA Welsh KA Plassman BL McDonald WM Helms MJ Anthony JC Neurology 1994 44 227 232 8309563 Anti-inflammatory drugs and Alzheimer disease McGeer PL McGeer E Rogers J Sibley J Lancet 1990 335 1037 10.1016/0140-6736(90)91101-F 1970087 Risk of Alzheimer's disease and duration of NSAID use Stewart WF Kawas C Corrada M Metter EJ Neurology 1997 48 626 632 9065537 Nonsteroidal anti-inflammatory drugs for the prevention of Alzheimer's disease: a systematic review Szekely CA Thorne JE Zandi PP Ek M Messias E Breitner JC Goodman SN Neuroepidemiology 2004 23 159 169 10.1159/000078501 15279021 Duration of nonsteroidal antiinflammatory drug use and risk of Alzheimer's disease. The Rotterdam study Veld BA Ruitenberg A Launer LJ Neurobiol Aging 2000 21 S204 Dynamic complexity of the microglial activation response in transgenic models of amyloid deposition: implications for Alzheimer therapeutics Morgan D Gordon MN Tan J Wilcock D Rojiani AM J Neuropathol Exp Neurol 2005 64 743 753 10.1097/01.jnen.0000178444.33972.e0 16141783 Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer's disease Lim GP Yang F Chu T Chen P Beech W Teter B Tran T Ubeda O Ashe KH Frautschy SA Cole GM J Neurosci 2000 20 5709 5714 10908610 Ibuprofen effects on Alzheimer pathology and open field activity in APPsw transgenic mice Lim GP Yang F Chu T Gahtan E Ubeda O Beech W Overmier JB Hsiao-Ashec K Frautschy SA Cole GM Neurobiol Aging 2001 22 983 991 10.1016/S0197-4580(01)00299-8 11755007 Deletion of tumor necrosis factor death receptor inhibits amyloid beta generation and prevents learning and memory deficits in Alzheimer's mice He P Zhong Z Lindholm K Berning L Lee W Lemere C Staufenbiel M Li R Shen Y J Cell Biol 2007 178 829 841 2064547 17724122 10.1083/jcb.200705042 Interferon-gamma and tumor necrosis factor-alpha regulate amyloid-beta plaque deposition and beta-secretase expression in Swedish mutant APP transgenic mice Yamamoto M Kiyota T Horiba M Buescher JL Walsh SM Gendelman HE Ikezu T Am J Pathol 2007 170 680 692 1851864 17255335 10.2353/ajpath.2007.060378 Activation of toll-like receptor 2 on microglia promotes cell up-take of Alzheimer's disease-associated amyloid beta peptide Chen K Iribarren P Hu J Chen J Gong W Cho EH Lockett S Dunlop NM Wang JM J Biol Chem 2006 281 3651 3659 10.1074/jbc.M508125200 16339765 CpG-containing oligodeoxynucleotide promotes microglial cell uptake of amyloid beta 1-42 peptide by up-regulating the expression of the G-protein- coupled receptor mFPR2 Iribarren P Chen K Hu J Gong W Cho EH Lockett S Uranchimeg B Wang JM FASEB J 2005 19 2032 2034 16219804 Microglial activation and amyloid-beta clearance induced by exogenous heat-shock proteins Kakimura J Kitamura Y Takata K Umeki M Suzuki S Shibagaki K Taniguchi T Nomura Y Gebicke-Haerter PJ Smith MA Perry G Shimohama S FASEB J 2002 16 601 603 11919167 Role of toll-like receptor signalling in A{beta} uptake and clearance Tahara K Kim HD Jin J Maxwell JA Li L Fukuchi KI Brain 2006 129 3006 3019 10.1093/brain/awl249 16984903 Intrahippocampal LPS injections reduce Abeta load in APP+PS1 transgenic mice DiCarlo G Wilcock D Henderson D Gordon M Morgan D Neurobiol Aging 2001 22 1007 1012 10.1016/S0197-4580(01)00292-5 11755009 Microglial activation is required for Abeta clearance after intracranial injection of lipopolysaccharide in APP transgenic mice Herber DL Mercer M Roth LM Symmonds K Maloney J Wilson N Freeman MJ Morgan D Gordon MN J Neuroimmune Pharmacol 2007 2 222 231 10.1007/s11481-007-9069-z 18040847 Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease Simard AR Soulet D Gowing G Julien JP Rivest S Neuron 2006 49 489 502 10.1016/j.neuron.2006.01.022 16476660 Bone-marrow-derived cells contribute to the recruitment of microglial cells in response to beta-amyloid deposition in APP/PS1 double transgenic Alzheimer mice Malm TM Koistinaho M Parepalo M Vatanen T Ooka A Karlsson S Koistinaho J Neurobiol Dis 2005 18 134 142 10.1016/j.nbd.2004.09.009 15649704 Innate immunity and transcription of MGAT-III and Toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin Fiala M Liu PT Espinosa-Jeffrey A Rosenthal MJ Bernard G Ringman JM Sayre J Zhang L Zaghi J Dejbakhsh S Chiang B Hui J Mahanian M Baghaee A Hong P Cashman J Proc Natl Acad Sci U S A 2007 104 12849 12854 1937555 17652175 10.1073/pnas.0701267104 Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease El Khoury J Toft M Hickman SE Means TK Terada K Geula C Luster AD Nat Med 2007 13 432 438 10.1038/nm1555 17351623 Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice Wyss-Coray T Yan F Lin AH Lambris JD Alexander JJ Quigg RJ Masliah E Proc Natl Acad Sci U S A 2002 99 10837 10842 125059 12119423 10.1073/pnas.162350199 TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice Wyss-Coray T Lin C Yan F Yu GQ Rohde M McConlogue L Masliah E Mucke L Nat Med 2001 7 612 618 10.1038/87945 11329064 Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase Jankowsky JL Fadale DJ Anderson J Xu GM Gonzales V Jenkins NA Copeland NG Lee MK Younkin LH Wagner SL Younkin SG Borchelt DR Hum Mol Genet 2004 13 159 170 10.1093/hmg/ddh019 14645205 Sustained hippocampal IL-1 beta overexpression mediates chronic neuroinflammation and ameliorates Alzheimer plaque pathology Shaftel SS Kyrkanides S Olschowka JA Miller JN Johnson RE O'Banion MK J Clin Invest 2007 117 1595 1604 1878531 17549256 10.1172/JCI31450 Pattern recognition receptors: doubling up for the innate immune response Gordon S Cell 2002 111 927 930 10.1016/S0092-8674(02)01201-1 12507420 Pathogen recognition and innate immunity Akira S Uematsu S Takeuchi O Cell 2006 124 783 801 10.1016/j.cell.2006.02.015 16497588 Toll-like receptors induce a phagocytic gene program through p38 Doyle SE O'Connell RM Miranda GA Vaidya SA Chow EK Liu PT Suzuki S Suzuki N Modlin RL Yeh WC Lane TF Cheng G J Exp Med 2004 199 81 90 1887723 14699082 10.1084/jem.20031237 Innate immune response gene expression profiles of N9 microglia are pathogen-type specific McKimmie CS Roy D Forster T Fazakerley JK J Neuroimmunol 2006 175 128 141 10.1016/j.jneuroim.2006.03.012 16697053 Broad expression of Toll-like receptors in the human central nervous system Bsibsi M Ravid R Gveric D van Noort JM J Neuropathol Exp Neurol 2002 61 1013 1021 12430718 Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs Olson JK Miller SD J Immunol 2004 173 3916 3924 15356140 Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay Opalka D Lachman CE MacMullen SA Jansen KU Smith JF Chirmule N Esser MT Clin Diagn Lab Immunol 2003 10 108 115 145272 12522048 10.1128/CDLI.10.1.108-115.2003 Inflammation in Alzheimer disease: driving force, bystander or beneficial response? Wyss-Coray T Nat Med 2006 12 1005 1015 16960575 The significance of neuroinflammation in understanding Alzheimer's disease Eikelenboom P Veerhuis R Scheper W Rozemuller AJ van Gool WA Hoozemans JJ J Neural Transm 2006 113 1685 1695 10.1007/s00702-006-0575-6 17036175 Neuroinflammation: implications for the pathogenesis and molecular diagnosis of Alzheimer's disease Rojo LE Fernandez JA Maccioni AA Jimenez JM Maccioni RB Arch Med Res 2008 39 1 16 10.1016/j.arcmed.2007.10.001 18067990 LPS receptor (CD14): a receptor for phagocytosis of Alzheimer's amyloid peptide Liu Y Walter S Stagi M Cherny D Letiembre M Schulz-Schaeffer W Heine H Penke B Neumann H Fassbender K Brain 2005 128 1778 1789 10.1093/brain/awh531 15857927 Toll-like receptors 2 and 4 mediate Abeta(1-42) activation of the innate immune response in a human monocytic cell line Udan ML Ajit D Crouse NR Nichols MR J Neurochem 2008 104 524 533 17986235 Role of the toll-like receptor 4 in neuroinflammation in Alzheimer's disease Walter S Letiembre M Liu Y Heine H Penke B Hao W Bode B Manietta N Walter J Schulz-Schuffer W Fassbender K Cell Physiol Biochem 2007 20 947 956 10.1159/000110455 17982277 Evidence for glial-mediated inflammation in aged APP(SW) transgenic mice Benzing WC Wujek JR Ward EK Shaffer D Ashe KH Younkin SG Brunden KR Neurobiol Aging 1999 20 581 589 10.1016/S0197-4580(99)00065-2 10674423 Beta-amyloid-induced glial expression of both pro- and anti-inflammatory cytokines in cerebral cortex of aged transgenic Tg2576 mice with Alzheimer plaque pathology Apelt J Schliebs R Brain Res 2001 894 21 30 10.1016/S0006-8993(00)03176-0 11245811 Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro apoptosis in healthy and Alzheimer's Disease (AD) individuals Lombardi VR Garcia M Rey L Cacabelos R J Neuroimmunol 1999 97 163 171 10.1016/S0165-5728(99)00046-6 10408971 The role of inflammation in Alzheimer's disease Tuppo EE Arias HR Int J Biochem Cell Biol 2005 37 289 305 10.1016/j.biocel.2004.07.009 15474976 Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages Harrington LE Hatton RD Mangan PR Turner H Murphy TL Murphy KM Weaver CT Nat Immunol 2005 6 1123 1132 10.1038/ni1254 16200070 Therapeutic efficacy of IL-17 neutralization in murine experimental autoimmune encephalomyelitis Hofstetter HH Ibrahim SM Koczan D Kruse N Weishaupt A Toyka KV Gold R Cell Immunol 2005 237 123 130 10.1016/j.cellimm.2005.11.002 16386239 Intrathecal activation of the IL-17/IL-8 axis in opticospinal multiple sclerosis Ishizu T Osoegawa M Mei FJ Kikuchi H Tanaka M Takakura Y Minohara M Murai H Mihara F Taniwaki T Kira J Brain 2005 128 988 1002 10.1093/brain/awh453 15743872 A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis Sutton C Brereton C Keogh B Mills KH Lavelle EC J Exp Med 2006 203 1685 1691 2118338 16818675 10.1084/jem.20060285 Production and functions of IL-17 in microglia Kawanokuchi J Shimizu K Nitta A Yamada K Mizuno T Takeuchi H Suzumura A J Neuroimmunol 2007 18164424 Co-expression of multiple transgenes in mouse CNS: a comparison of strategies Jankowsky JL Slunt HH Ratovitski T Jenkins NA Copeland NG Borchelt DR Biomol Eng 2001 17 157 165 10.1016/S1389-0344(01)00067-3 11337275 Amyloid beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease Verdier Y Zarandi M Penke B J Pept Sci 2004 10 229 248 10.1002/psc.573 15160835