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. 2015 Aug 9:12:144.
doi: 10.1186/s12974-015-0339-z.

Pharmacological antagonism of interleukin-8 receptor CXCR2 inhibits inflammatory reactivity and is neuroprotective in an animal model of Alzheimer's disease

Jae K Ryu  1 , T Cho  2 , Hyun B Choi  3 , N Jantaratnotai  4 , James G McLarnon  5
Affiliations

Pharmacological antagonism of interleukin-8 receptor CXCR2 inhibits inflammatory reactivity and is neuroprotective in an animal model of Alzheimer's disease

Jae K Ryu et al. J Neuroinflammation. .

Abstract

Background: The chemokine interleukin-8 (IL-8) and its receptor CXCR2 contribute to chemotactic responses in Alzheimer's disease (AD); however, properties of the ligand and receptor have not been characterized in animal models of disease. The primary aim of our study was to examine effects of pharmacological antagonism of CXCR2 as a strategy to inhibit receptor-mediated inflammatory reactivity and enhance neuronal viability in animals receiving intrahippocampal injection of amyloid-beta (Aβ1-42).

Methods: In vivo studies used an animal model of Alzheimer's disease incorporating injection of full-length Aβ1-42 into rat hippocampus. Immunohistochemical staining of rat brain was used to measure microgliosis, astrogliosis, neuronal viability, and oxidative stress. Western blot and Reverse Transcription PCR (RT-PCR) were used to determine levels of CXCR2 in animal tissue with the latter also used to determine expression of pro-inflammatory mediators. Immunostaining of human AD and non-demented (ND) tissue was also undertaken.

Results: We initially determined that in the human brain, AD relative to ND tissue exhibited marked increases in expression of CXCR2 with cell-specific receptor expression prominent in microglia. In Aβ1-42-injected rat brain, CXCR2 and IL-8 showed time-dependent increases in expression, concomitant with enhanced gliosis, relative to controls phosphate-buffered saline (PBS) or reverse peptide Aβ42-1 injection. Administration of the competitive CXCR2 antagonist SB332235 to peptide-injected rats significantly reduced expression of CXCR2 and microgliosis, with astrogliosis unchanged. Double staining studies demonstrated localization of CXCR2 and microglial immunoreactivity nearby deposits of Aβ1-42 with SB332235 effective in inhibiting receptor expression and microgliosis. The numbers of neurons in granule cell layer (GCL) were reduced in rats receiving Aβ1-42, compared with PBS, with administration of SB332235 to peptide-injected animals conferring neuroprotection. Oxidative stress was indicated in the animal model since both 4-hydroxynonenal (4-HNE) and hydroethidine (HEt) were markedly elevated in Aβ1-42 vs. PBS-injected rat brain and diminished with SB332235 treatment.

Conclusion: Overall, the findings suggest critical roles for CXCR2-dependent inflammatory responses in an AD animal model with pharmacological modulation of the receptor effective in inhibiting inflammatory reactivity and conferring neuroprotection against oxidative damage.

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Figures

Fig. 1
Fig. 1
Staining patterns of CXCR2 in AD and ND cortical and hippocampal brain sections. a Representative CXCR2 immunoreactivity (ir) in cortical regions of ND and AD brain; scale bar represents 40 μm. b Quantification of CXCR2 area density in ND and AD sections (N = 6 cases for each); asterisk denotes p < 0.05. c Double staining of CXCR2 (green), HLA-DR-(+)ve microglia (red) and merged CXCR2/HLA-DR in cortical AD brain. d Double staining for the same markers in hippocampal brain sections; scale bar for c (and d, is same as c) is 100 μm
Fig. 2
Fig. 2
Expression of CXCR2 and IL-8 in ML region of rat dentate gyrus. a Representative RT-PCR for CXCR2 and IL-8 in controls (3 days post-injection of PBS or reverse peptide Aβ42–1) and in Aβ1–42-injected rat brain (1, 3, and 7 days post-injection); β-actin was used as a reaction standard. b Semi-quantification of RT-PCR for CXCR2 (left bar graph) and IL-8 (right bar graph); N = 5 animals per treatment group. c Typical CXCR2 ir for PBS, Aβ42–1, and Aβ1–42 (3 days post-injection); scale bar is for 70 μm. d Overall CXCR2 area density for the different animal groups (N = 4 animals per treatment group). Asterisk denotes p < 0.05 for Aβ1–42 vs PBS. e Representative Western blot for CXCR2 in control (3 days) and 1,3, and 7 days post-peptide injection. The bar graph shows relative CXCR2 levels for control and different durations of peptide injection (N = 4 animals per group)
Fig. 3
Fig. 3
Cell-specific expression of CXCR2 in ML of dentate gyrus. a Representative single and merged staining of OX-42-(+)ve microglia with CXCR2 at 3 days post-Aβ1–42 intrahippocampal injection; scale bar is for 20 μm. b Single and merged staining of GFAP-(+)ve astrocytes with CXCR2 after 3 days of peptide injection; scale bar is for 15 μm
Fig. 4
Fig. 4
Effects of SB332235 on gliosis in ML region of dentate gyrus in peptide-injected hippocampus. a Representative microgliosis (Iba-1 marker) following 3 days injections with PBS (upper left panel), SB332235 alone (upper right panel), Aβ1–42 (lower left panel), and Aβ1–42 + SB332235 (lower right panel). b Overall area density for Iba-1 (N = 5 per treatment group). c Representative astrogliosis (GFAP marker) for PBS (upper left panel), SB332235 alone (upper right panel), Aβ1–42 (lower left panel), and Aβ1–42 + SB332235 (lower right panel). d Overall area density for GFAP (N = 5 per treatment group). Scale bars are for 80 μm. *p < 0.05 Aβ1–42 vs PBS; #p < 0.05 Aβ1–42 + SB332235 vs Aβ1–42
Fig. 5
Fig. 5
Effects of SB332235 on area density for CXCR2 and microglia and astrocyte responses in proximity to peptide deposits in ML region. a Representative CXCR2 ir nearby Aβ1–42 (3 days post-Aβ1–42 injection) in the absence (left panel) and presence (right panel) of SB332235 treatment; scale bar is for 50 μm. b Overall CXCR2 area density (N = 5 per group) in a single quadrant within 300 μm of peptide. c Representative Iba-1 ir nearby Aβ1–42 in the absence (left panel) and presence (right panel) of SB332235 treatment; scale bar is for 30 μm. d Overall Iba-1 area density (N = 5 per group) in regions within 300 μm of peptide. e Representative GFAP ir nearby peptide deposits in the absence (left panel) and presence of SB332235 (right panel); scale bar is for 30 μm. f Overall GFAP area density (N = 5 per group) within 300 μm of peptide. *p < 0.05 for Aβ1–42 vs Aβ1–42 + SB332235
Fig. 6
Fig. 6
Neuroprotective and lipid peroxidation effects of SB332235 on GCL neurons. a Representative neuronal staining (NeuN) in PBS control (upper left panel), SB332235 alone (upper right panel), Aβ1–42 (lower left panel), and Aβ1–42 + SB332235 (lower right panel); results are for 3 days post-injection; scale bar represents 50 μm. b Area density of NeuN for the animal groups, N = 5 per group. Asterisk denotes p < 0.05. c Typical lipid peroxidation (4-HNE marker) levels following 3 days intrahippocampal injection of PBS (upper left panel), SB332235 alone (upper right panel), Aβ1–42 (lower left panel), and Aβ1–42 + SB332235 (lower right panel). Scale bar is for 50 μm. d Area density of 4-HNE for the different animal treatments, N = 5 per group. *p < 0.05 for Aβ1–42 vs PBS and #p < 0.05 for Aβ1–42 vs Aβ1–42 + SB332235
Fig. 7
Fig. 7
Effects of SB332235 on superoxide activity and inflammatory factors. a Representative superoxide ir (HEt) in region adjacent to GCL after 3 days intrahippocampal injection of PBS (upper left panel), SB332235 alone (upper right panel), Aβ1–42 (lower left panel), and Aβ1–42 + SB332235 (lower right panel); scale bar is for 120 μm. b Quantification of intensity of HEt for the different animal groups; N = 5 per group; *p < 0.05 for Aβ1–42 vs PBS and #p < 0.05 for Aβ1–42 vs Aβ1–42 + SB332235
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