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Pharmacological antagonism of interleukin-8 receptor CXCR2 inhibits inflammatory reactivity and is neuroprotective… Ryu, Jae K; Cho, T; Choi, Hyun B; Jantaratnotai, N; McLarnon, James G Aug 9, 2015

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RESEARCH Open AccessPharmacological antagonism of interleukin-8since both 4-hydroxynonenal (4-HNE) and hydroethidine (HEt) were markedly elevated in Aβ1–42 vs PBS-injected rat brainnferringinjection,JOURNAL OF NEUROINFLAMMATIONRyu et al. Journal of Neuroinflammation  (2015) 12:144 DOI 10.1186/s12974-015-0339-zV6T 1Z3, CanadaFull list of author information is available at the end of the article* Correspondence: mclarnon@mail.ubc.ca1Department of Anesthesiology, Pharmacology and Therapeutics, Universityof British Columbia, 2176 Health Science Mall, Vancouver, British Columbiamodel with pharmacological modulation of the receptor effective in inhibiting inflammatory reactivity and coneuroprotection against oxidative damage.Keywords: CXCR2, CXCR2 antagonist, SB332235, Interleukin-8 (IL-8), Amyloid-beta (Aβ1–42) intrahippocampalAlzheimer’s disease (AD), AD animal model, Microgliosis, Neuroprotection, Oxidative damageand diminished with SB332235 treatment.Conclusion: Overall, the findings suggest critical roles for CXCR2-dependent inflammatory responses in an AD animalreceptor CXCR2 inhibits inflammatoryreactivity and is neuroprotective in ananimal model of Alzheimer’s diseaseJae K Ryu1, T Cho2, Hyun B Choi2, N Jantaratnotai3 and James G McLarnon1*AbstractBackground: The chemokine interleukin-8 (IL-8) and its receptor CXCR2 contribute to chemotactic responses inAlzheimer’s disease (AD); however, properties of the ligand and receptor have not been characterized in animalmodels of disease. The primary aim of our study was to examine effects of pharmacological antagonism of CXCR2as a strategy to inhibit receptor-mediated inflammatory reactivity and enhance neuronal viability in animalsreceiving 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 intorat hippocampus. Immunohistochemical staining of rat brain was used to measure microgliosis, astrogliosis, neuronalviability, and oxidative stress. Western blot and Reverse Transcription PCR (RT-PCR) were used to determine levelsof 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 inexpression 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 tocontrols phosphate-buffered saline (PBS) or reverse peptide Aβ42–1 injection. Administration of the competitiveCXCR2 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 immunoreactivitynearby deposits of Aβ1–42 with SB332235 effective in inhibiting receptor expression and microgliosis. The numbers ofneurons in granule cell layer (GCL) were reduced in rats receiving Aβ1–42, compared with PBS, with administration ofSB332235 to peptide-injected animals conferring neuroprotection. Oxidative stress was indicated in the animal model© 2015 Ryu et al. Open Access This article is dInternational License (http://creativecommonsreproduction in any medium, provided you gthe Creative Commons license, and indicate if(http://creativecommons.org/publicdomain/zeistributed under the terms of the Creative Commons Attribution 4.0.org/licenses/by/4.0/), which permits unrestricted use, distribution, andive appropriate credit to the original author(s) and the source, provide a link tochanges were made. The Creative Commons Public Domain Dedication waiverro/1.0/) applies to the data made available in this article, unless otherwise stated.cases. The ND cases exhibited no clinical or pathologicalmounted on glass slides and coverslipped with ProlongRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 2 of 13BackgroundChronic inflammation is an inherent ongoing processin the progression of Alzheimer’s disease [1–3]. How-ever, the specific mechanisms by which sustained in-flammatory reactivity contributes to the progressiveneuronal degeneration underlying loss of cognition inAlzheimer’s disease (AD) brain are not well under-stood. Some evidence suggests limited benefits of non-steroidal anti-inflammatory drugs (NSAIDS) [4] withthe relatively small extent of drug efficacy attributedto previous deterioration in cognitive function in ADindividuals prior to medication. Another possibility isthat inflammatory reactivity in AD brain is manifestfrom activation of multiple pathways other thancyclooxygenase-dependent activity targeted by NSAIDS.A critical component of inflammatory response is achemokine-mediated mobilization of microglia in re-sponse to peptide deposition [3, 5–7]. A spectrum ofchemokines contributes to inflammatory responses indisease [8, 9], with some evidence suggesting a promin-ent role for interleukin-8 (IL-8) in AD pathology. Genemicroarray analysis has shown that IL-8 exhibits the lar-gest increase in expression of any inflammatory factor inhuman microglia incubated with amyloid-beta (Aβ1–42)[10]. This same group also reported dose-dependent in-creases in production of IL-8 in human microglia stimu-lated with peptide [11]. Elevated cerebrospinal fluid (csf)levels of IL-8 have been documented in AD brain rela-tive to controls [12]. Interestingly, IL-8 has been re-ported to potentiate Aβ1–42-induced expression andproduction of a number of pro-inflammatory cytokinesin cultured human microglia [13].Immunostaining for the IL-8 receptor CXCR2 hasdemonstrated receptor association with neuritic pla-ques in AD tissue [5, 14]. However, CXCR2 alsoransduces IL-8-dependent cellular inflammatory che-mokine responses in the periphery and brain. In theformer case, the receptor is expressed by infiltratingneutrophils in chronic obstructive pulmonary disease(COPD) with inhibition of CXCR2-mediated inflam-matory responses effective in attenuating lung damage[15]. Prominent CXCR2 activity in activated microgliahas been reported in damaged brain with antagonismof receptor effective in reducing inflammation andpromoting recovery in lesioned spinal cord [16], fol-lowing traumatic brain insult [17] and in animal tumormodels [18].At present, pharmacological modulation of CXCR2has not been examined in animal models of AD. Weposited that given the high levels of IL-8 in AD brainthat pharmacological inhibition of CXCR2 could serveas a novel strategy to protect neurons exposed to inflam-matory microenvironments. To examine this hypothesis,we have used the compound SB332235, a selectiveGold anti-fading agent (Invitrogen). For double-immunofluorescence staining [23], free-floating sec-tions were incubated for 48 h at 4 °C with a mixture oftwo primary antibodies: CXCR2/HLA-DR and CXCR2/GFAP. After incubation with the indicated primaryantibodies, sections were rinsed in PBST and incubatedfor 1 h at room temperature with a mixture of AlexaFluor 488 goat anti-rabbit IgG (1:200; Invitrogen) andhistory of dementia or other neurological disorders. Fiveof the ND cases were scored as Braak stage I with onecase scored as Braak stage II [21]. All cases of AD met theclinical criteria and postmortem confirmation for AD [22]and were characterized by high levels of plaque densityand neurofibrillary tangles. The AD cases were rated asBraak V (one case) or VI (five cases).Immunohistochemical staining and analysis in human NDand AD sectionsFor immunofluorescent staining, free-floating sections(30 μm) from ND and AD tissues were washed inphosphate-buffered saline (PBS) with Triton X-100(PBST; 0.01 M PBS, pH 7.4, containing 0.3 % Triton X-100) and transferred into 5 % skim milk in PBST for 1 h.Sections were then incubated for 48 h at 4 °C with anti-bodies for CXCR2, HLA-DR, or GFAP and then rinsedin PBST and incubated with Alexa Fluor 488-conjugatedgoat anti-rabbit IgG (1:200; Invitrogen) for 1 h at roomtemperature. After washing in PBST, sections wereinhibitor of CXCR2 in macrophage cells [15, 19], as a re-ceptor antagonist to attenuate microglial inflammatoryreactivity induced by Aβ1–42 intrahippocampal injection.Specifically, SB332235 has been examined in vivo as amodulator of CXCR2 cell-specific association, gliosis,microglial chemotactic response, oxidative stress factors,and neuronal viability.MethodsHuman brain tissuePreparation of human ND and AD sectionsThe procedures used to isolate postmortem tissue havebeen described [20]. Entorhinal cortical sections fromsix ND cases (ages from 60 to 85 years, postmortem in-tervals, 6–24 h) and six AD cases (ages from 64 to87 years, postmortem intervals, 5–10 h) were obtainedfrom the Kinsmen Laboratory brain bank at the Universityof British Columbia (UBC, Vancouver, British Columbia,Canada). Average age of individuals and mean postmor-tem delay did not differ significantly between AD and NDAlexa Fluor 594 goat anti-mouse IgG secondary anti-body (1:200; Invitrogen).out for microglial and astrocytic CXCR2 expression. InRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 3 of 13In vivo studies using intrahippocampal injection of AβpeptideSurgical procedures. All animal procedures were ap-proved by the UBC Animal Care Ethics Committee, withadherence to guidelines of the Canadian Council onAnimal Care. Male Sprague Dawley rats (Charles RiverLaboratories, Montreal, QC, Canada) weighing 280–300 g were used for in vivo studies. In brief, rats wereinjected intraperitoneal (ip) with an anesthetic mixtureof ketamine hydrochloride (100 mg/kg; Bimeda-MTC,Cambridge, ON, Canada) and xylazine hydrochloride(10 mg/kg; Bayer Inc., Etobicoke, ON, Canada) and wereplaced in a stereotaxic apparatus (David Kopf Instru-ments, Tujunga, CA, USA). Animals received stereotaxicinjection of Aβ1–42 or controls (PBS or reverse peptideAβ42–1) as previously described [6, 24–26]. Followingskin incision to expose the skull, peptides (CaliforniaPeptides, Napa, CA, USA) were slowly injected (0.2 μl/min) into the dentate gyrus region of rat hippocampus.Injection coordinates were as follows: anterior-posterior(AP), −3.3 mm; medial-lateral (ML), −1.6 mm; dorsoven-tral (DV), −3.2 mm; all measurements from bregma.Preparation and administration of chemicalsAmyloid peptide. The procedures for preparation ofamyloid-beta peptide for intrahippocampal injectionhave been described [6, 25, 26]. Full-length Aβ1–42 or re-verse peptide Aβ42–1 (California Peptide, Napa, CA,USA) was first dissolved in 35 % acetonitrile (Sigma) andfurther diluted to 500 μM with incremental additions ofPBS with vortexing. The peptide solution was subse-quently incubated at 37 °C for 18 h to promote fibrilliza-tion and aggregation and stored at 20 °C [11, 24].Peptides (2 nmol) were injected for durations of 1, 3,and 7 days in this work.SB332235. This compound was kindly donated byGlaxoSmithKline (709 Swedeland Road, King of Prussia,PA, USA). The compound was dissolved in a saline solu-tion and applied by ip injection at a single dose of 1 mg/kg at the time of peptide injection. SB332235 has beencharacterized as a specific antagonist for CXCR2-mediated functional responses [15, 27].Immunohistochemical staining of rat brainAnimals were transcardially perfused with heparinizedcold saline followed by 4 % paraformaldehyde underketamine/xylazine anesthesia. Brains were then removed,postfixed, cryoprotected, and cut into 40-μm sections[6]. Free-floating sections were processed for immuno-histochemistry as described previously [6, 24, 26].Briefly, sections were incubated in PBS containing 1 %bovine serum albumin, normal goat serum (NGS), and0.2 % Triton X-100 (Sigma-Aldrich, St Louis, MO, USA)for 1 h. Sections were incubated overnight at 4 °C withthe former case, since Iba-1 antibody was raised inrabbit, mouse OX-42 (1:500; Serotec, Oxford, UK) wasused for staining of receptor in microglia. CXCR2 asso-ciation with astrocytes used respective antibodies for re-ceptor/cell of CXCR2/GFAP. Sections were rinsed inPBS with 0.5 % BSA and incubated with a mixture ofsecondary antibodies (Alexa Fluor 488 and 594; 1:100;Invitrogen).To determine production of reactive oxygen species(ROS), peptide-injected animals received ip injection of1 mg/kg hydroethidine (HEt; Molecular Probes) which isoxidized to ethidium bromide in the presence of super-oxide radicals [28]. At 3 h following HEt injection, animalswere killed by transcardiac saline perfusion and brainswere removed and frozen. Coronal sections (40-μm thick-ness) of hippocampus were examined under a Zeiss Axio-plan 2 fluorescent microscope equipped with an ethidiumfilter and digital video camera (DVC) system (DiagnosticInstruments, Sterling Heights, MI, USA).Immunohistochemical analysis of rat brainQuantification of immunohistochemical staining followedpublished procedures [25, 26, 29]. Digitized images wereobtained with a Zeiss Axioplan 2 fluorescent microscopeequipped with a DVC system. Quantitative image analysisfor the immunostained rat hippocampal sections was per-formed on three equally spaced sections through the levelof the injection site. In each stained section, hippocampalboundaries were outlined with the granule cell layer(GCL) denoted as the superior blade of dentate gyrus.The molecular layer (ML) was then defined as the re-gion between GCL border and hippocampal fissure.Neuronal viability and lipid peroxidation were mea-sured in GCL, and glial responses and superoxide pro-duction were measured in adjacent ML. Digitizedthe following primary antibodies: anti-glial fibrillaryacidic protein, a marker for astrocytes (GFAP; 1:1000;Sigma-Aldrich), anti-neuronal nuclei (NeuN; 1:500;Chemicon, Temecula, CA, USA), and two specific micro-glial antibodies (anti-ionized calcium-binding adaptermolecule 1 (Iba-1; 1:500; Wako Chemicals, Richmond VA,USA) and HLA-DR (1:1000; Dako, Mississauga, ON,Canada). Other antibodies used included ones for CXCR2(1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA),Aβ1–42 (1:100; Dako), and 4-hydroxynonenal (4-HNE,1:500 Jaica, Shizuoka, Japan). Sections were rinsed in PBSwith 0.5 % BSA and incubated with secondary antibodiesconjugated with Alexa Fluor 488 or 594 (1:200; Invitrogen,Burlington, ON, Canada) for 1 h in the dark.In this work, double immunostaining was also carriedimages were analyzed using Northern Eclipse software(Empix Imaging, Mississauga, ON, Canada).26, 30, 31] was used to characterize glial reactivity andRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 4 of 13RT-PCR in peptide-injected rat hippocampusThe specific protocols for Reverse Transcription PCR (RT-PCR) closely followed those outlined in previous work fromthis laboratory [26, 29, 30]. Anesthetized animals werekilled by decapitation at 1, 3, and 7 days after peptide injec-tion. The control animals were killed at 3 days after PBS orreverse peptide Aβ42–1 injection. Brains were removed, andhippocampal tissues were freshly dissected onto cold metaltissue matrices (Harvard Apparatus) and quickly frozen inliquid nitrogen. Total RNA was extracted using Trizol re-agent (Invitrogen) and processed using reverse transcript-ase; cDNA products were amplified by PCR using aGeneAmp thermal cycler (Applied Biosystems, Foster City,CA, USA) with Taq polymerase. PCR primers (β-actin wasused as a reaction control) were as follows: CXCR2: for-ward, 5′-GTC AGG ATC CAA GTT TAC CTC AAAAAT GG-3′; reverse, 5′-CTT AGG TCG ACG GTC TTAGAG AGT AGT GG-3′. The primers for IL-8 were as fol-lows: forward, 5′-ACT GAG AGT GAT TGA GAG TGGAC AC-3′; reverse 5′-AAC CCT CTG CAC CCA GTTTTC-3′. Relative mRNA levels (stimulated values normal-ized to controls) were obtained using NIH ImageJ software1.24 (National Institute of Health, Bethesda, MD, USA).Western blot for CXCR2Total protein from rat hippocampal tissue was used forWestern blot analysis. Protein samples (50 μg) were sub-jected to SDS-PAGE prior to transfer onto a PVDF mem-brane (Millipore, Bedford, MA, USA), blocked with either5 % skim milk or bovine serum albumin, and probed withanti-CXCR2 (1:200; Santa Cruz Biotechnology) and β-actin(1:5000; Abcam, Cambridge, MA, USA). HRP-conjugatedsecondary antibodies (GE Healthcare biosciences, Piscat-away, NJ, USA) were used to develop immunoblots whichwere processed using enhanced chemiluminescence (ECL)detection (GE Healthcare Biosciences). Band intensitieswere quantified using ImageJ software (NIH).Statistical analysisResults are presented as mean ± SEM. The statisticalanalysis was performed using a one-way ANOVA,followed by the Student–Newman–Keuls multiple com-parison test or Student’s t test (GraphPad Prism 3.0;Graph Pad) with significance level set at p < 0.05.ResultsCXCR2 expression in AD and ND brain sectionsBrain tissue from AD and ND individuals was first ana-lyzed for expression of CXCR2. Representative immuno-staining demonstrated low levels of CXCR2 in areas ofentorhinal cortex from ND tissue with a considerably el-evated expression of the IL-8 receptor in AD sections(Fig. 1a). Quantification for CXCR2 expression is pre-sented in Fig. 1b (N = 6 for each of AD/ND). The areaneuronal viability and their pharmacological modula-tions with the CXCR2 antagonist, SB332235. Initial ex-periments examined the expression of CXCR2 and aligand for the receptor, IL-8 at different durations fol-lowing peptide injection.Representative RT-PCR for CXCR2, and also for IL-8,is shown for durations of Aβ1–42 injections of 1, 3, and7 days (Fig. 2a). Controls used both PBS and reversepeptide (Aβ42–1) with results shown at a single timepoint of 3 days post-injection. The results showed levelsof both CXCR2 and IL-8 were higher at all time pointspost-Aβ1–42 injection compared with 3 days controls(PBS and Aβ42–1). Interestingly, both CXCR2 and ligandIL-8 were maximally expressed at 3 days following pep-tide injection.Semi-quantification of data for CXCR2 expression (N =5 animals/group) for the different peptide injection timesis shown (Fig. 2b). Expression of CXCR2 (left bar graph)was increased with Aβ1–42 by respective amounts of 1.4-fold, 3.1-fold, and 1.9-fold (1, 3 and 7 days Aβ1–42) com-pared with 3 days PBS injection; the 3 and 7 days valuesrepresenting significant increases. The corresponding datafor IL-8 are presented in Fig. 2b (right bar graph) with ex-pression of the chemokine (N = 5 animals/group) in-density of CXCR2 was increased 4.4-fold in AD, com-pared with ND, brain tissue.Since the focus of the study was pharmacologicalmodulation of CXCR2-mediated inflammatory reactivityin vivo, we also examined receptor expression and themicroglial marker HLA-DR in AD brain. Representativesingle and double staining for CXCR2/HLA-DR in cor-tical brain sections are presented in Fig. 1c. The resultsdemonstrated considerable co-localization for the twomarkers, a similar finding was made in all AD cases.Typical expression of CXCR2/HLA-DR in AD hippo-campal brain tissue is shown in Fig. 1d. Both markersshowed a marked extent of co-localization throughoutareas of hippocampus. Although hippocampal tissue waslimited in availability, similar co-localization between re-ceptor and HLA-DR was evident in sections from N = 3other AD cases. Although we did not attempt quantifica-tion for overall merged staining for CXCR2/HLA-DR,the findings from AD cortical and hippocampal tissueimplicated microglial CXCR2 as a putative inflammatorymediator in the progression of AD pathology. Experi-ments were then designed to examine effects of pharma-cological antagonism of CXCR2 in animal brain.Time-dependent CXCR2 expression in vivoAn Aβ1–42 intrahippocampal-injection animal model [6,creased by 3.3-fold, 6-fold, and 4-fold with peptideinjections (respective values for 1, 3, and 7 daysRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 5 of 13application) compared with PBS 3 days injection; all valuesrepresent significant increases.Additional experiments focused on CXCR2 expressionin peptide-injected hippocampus. We used immunohis-tochemical staining in the ML of dentate gyrus tomeasure CXCR2 levels at the single time point of 3 dayspost-Aβ1–42 injection. As shown in Fig. 2c, relatively lowlevels of CXCR2 immunoreactivity (ir) were evident witheither PBS (left panel) or reverse peptide (middle panel)controls. However, Aβ1–42-injected animals exhibited amarked increase in CXCR2 expression (right panel) withassociation of receptor in cells showing a glial morph-ology. Quantification of immunostaining data is pre-sented in Fig. 2d (N = 5 animals/group). The area densityof CXCR2 was considerably increased (by 6.3-fold) withAβ1–42, compared with PBS, injection. Thus, upregula-tion of CXCR2 expression is a characteristic response topeptide injection in the AD animal model.Western blot analysis was done to demonstrate pro-tein expression of CXCR2. As shown in Fig. 2e, CXCR2was minimally expressed in control, progressively in-creased at 1 and 3 days post-intrahippocampal injectionof Aβ1–42 and returned to near control level at 7 daysfollowing peptide injection. Quantification of CXCR2levels (N = 4 animals per group) showed that at 1 dayFig. 1 Staining patterns of CXCR2 in AD and ND cortical and hippocampalregions of ND and AD brain; scale bar represents 40 μm. b Quantification oasterisk denotes p < 0.05. c Double staining of CXCR2 (green), HLA-DR-(+)ved Double staining for the same markers in hippocampal brain sections; scapost-Aβ1–42 injection, receptor expression was elevatedbut not significantly different from control. CXCR2 wasmaximally expressed at 3 days post-peptide and signifi-cantly increased (by 40 %) from levels with PBSinjection.Cell-specific expression of CXCR2 in vivoThe patterns of CXCR2 immunostaining shown in Fig. 1for human AD tissue indicated prominent receptor ex-pression in microglia. Double immunostaining was usedto study glial-dependent expression of CXCR2 in controland peptide-injected rat hippocampus. At maximalCXCR2 expression (3 days of Aβ1–42 injection), doublestaining was carried out to determine association of IL-8receptor with microglia (OX-42 marker) and astrocytes(GFAP marker). As for previous studies on gliosis in theAD animal model, gliosis was measured in the ML re-gion of the dentate gyrus. This procedure would serve tominimize contributions from receptor expression inneurons.Representative patterns of immunostaining are shownfor OX-42-(+)ve microglia and CXCR2 in Fig. 3a (3 dayspost-Aβ1–42 injection). Considerable association of thetwo markers was evident in the merged staining (rightpanel) with results indicating marked Aβ1–42 stimulationbrain sections. a Representative CXCR2 immunoreactivity (ir) in corticalf CXCR2 area density in ND and AD sections (N = 6 cases for each);microglia (red) and merged CXCR2/HLA-DR in cortical AD brain.le bar for c (and d, is same as c) is 100 μmRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 6 of 13of microgliosis. Typical astrocytic staining (GFAP) withCXCR2 is presented in Fig. 3b. Merged staining indi-cated that a relatively low proportion of astrocytes wereco-localized with CXCR2 (right panel, Fig. 3b).The immunostaining results with both OX-42 andGFAP suggested considerable gliosis was induced inpeptide-injected rat hippocampus which was examinedas a target for pharmacological modulation of CXCR2using the receptor antagonist, SB332235.Effects of CXCR2 antagonist SB332235 on gliosisInitial experiments were designed to examine effects ofCXCR2 antagonism, at a single time point of 3 dayspost-peptide injection, on microgliosis and astrogliosis.Sections were isolated from the ML region of hippo-campus to minimize neuronal expression of receptor.Animal groups received PBS and reverse peptideFig. 2 Expression of CXCR2 and IL-8 in ML region of rat dentate gyrus. a Rinjection of PBS or reverse peptide Aβ42–1) and in Aβ1–42-injected rat brainstandard. b Semi-quantification of RT-PCR for CXCR2 (left bar graph) and IL-ir for PBS, Aβ42–1, and Aβ1–42 (3 days post-injection); scale bar is for 70 μm.animals per treatment group). Asterisk denotes p < 0.05 for Aβ1–42 vs PBS. eand 7 days post-peptide injection. The bar graph shows relative CXCR2 levanimals per group)controls, Aβ1–42, Aβ1–42 with SB332235 treatment andSB332235 alone.Representative microglial ir (Iba-1 marker) is shown inFig. 4a and indicates relatively low numbers of cells inPBS control (upper left panel) and with SB332235 ap-plied alone (upper right panel). Considerable microglio-sis was induced following peptide injection (lower leftpanel). Treatment of peptide-injected animals withSB332235 was effective in attenuating microglial re-sponses (lower right panel).Quantification of data is presented in Fig. 4b (N = 5animals/group) and also includes reverse peptide Aβ42–1as a control animal group. Both PBS and reverse peptidedemonstrated similar low values of microglial Iba-1 ir.Iba-1 ir, used as an index of microgliosis, was increased3.5-fold in Aβ1–42, compared with PBS, injected ratbrain. Peptide-administered animals receiving SB332235epresentative RT-PCR for CXCR2 and IL-8 in controls (3 days post-(1, 3, and 7 days post-injection); β-actin was used as a reaction8 (right bar graph); N = 5 animals per treatment group. c Typical CXCR2d Overall CXCR2 area density for the different animal groups (N = 4Representative Western blot for CXCR2 in control (3 days) and 1,3,els for control and different durations of peptide injection (N = 4exhibited significantly reduced microgliosis, by 45 %,compared with rats receiving Aβ1–42 in the absence ofthe CXCR2 antagonist. Similar levels of Iba-1 ir weremeasured with SB332235 administered alone and withPBS and Aβ42–1 controls.Typical astrocytic staining (GFAP marker) indicated aconsiderably enhanced response in Aβ1–42-injected ratsrelative to PBS controls (Fig. 4c, left panels). Interest-ingly, unlike the results for microglia, astrogliosisremained elevated with SB332235 treatment of peptide-injected animals (lower right panel). GFAP stainingwith SB332235 applied separately (upper right panel)was similar to PBS control. Overall (N = 5 animals/group), peptide-injected hippocampus demonstrated anincreased astrogliosis (by 3.5-fold) compared with PBS(Fig. 4d). In the presence of SB332235 application withAβ1–42, GFAP ir was reduced by 14 %, an insignificantchange compared with no drug treatment. Levels ofGFAP were not significantly different between animalsreceiving SB332235 treatment and PBS or reverse pep-tide controls.Fig. 3 Cell-specific expression of CXCR2 in ML of dentate gyrus. aRepresentative single and merged staining of OX-42-(+)ve microgliawith CXCR2 at 3 days post-Aβ1–42 intrahippocampal injection; scale baris for 20 μm. b Single and merged staining of GFAP-(+)ve astrocyteswith CXCR2 after 3 days of peptide injection; scale bar is for 15 μmFig. 4 Effects of SB332235 on gliosis in ML region of dentate gyrus in peptfollowing 3 days injections with PBS (upper left panel), SB332235 alone (uppright panel). b Overall area density for Iba-1 (N = 5 per treatment group). cSB332235 alone (upper right panel), Aβ1–42 (lower left panel), and Aβ1–42 + Streatment group). Scale bars are for 80 μm. *p < 0.05 Aβ1–42 vs PBS; #p < 0.0Ryu et al. Journal of Neuroinflammation  (2015) 12:144 Page 7 of 13ide-injected hippocampus. a Representative microgliosis (Iba-1 marker)er right panel), Aβ1–42 (lower left panel), and Aβ1–42 + SB332235 (lowerRepresentative astrogliosis (GFAP marker) for PBS (upper left panel),B332235 (lower right panel). d Overall area density for GFAP (N = 5 per5 Aβ1–42 + SB332235 vs Aβ1–42Effects of SB332235 on CXCR2 expression, gliosis, andmicroglial chemotaxis nearby Aβ1–42 depositsPrevious work has indicated microglial chemotactic re-sponses in the Aβ1–42 injection animal model as an ini-tial inflammatory response to deposition of peptide [6,7]. The consequence of this rapid response is the spatiallocalization of microgliosis and possibly upregulatedCXCR2 ir in the vicinity of peptide deposits in the MLlayer of dentate gyrus. Experiments were designed toexamine chemotaxis in vivo and to measure SB332235modulation of receptor expression and gliosis nearbyamyloid deposits.We determined immunoreactivities of CXCR2, Iba-1,and GFAP within 300 μm of Aβ deposits in the ML re-gion. The procedure defined quadrants of ML regionswith a focal point denoted by Aβ plaque deposition.Representative staining for CXCR2 in proximity topeptide is shown in the absence (left column) and pres-ence (right column) of SB332235 treatment of peptide-injected (3 days) animals (Fig. 5a). The distribution ofCXCR2 ir was concentrated nearby Aβ deposits inpeptide-injected hippocampus (left panel, Fig. 5a) withSB332235 effective in reducing receptor expressionwhen administered with peptide (right panel, Fig. 5a).Overall (N = 5 animals/group), CXCR2 expression inproximity to Aβ was diminished by 45 % with applica-tion of SB332235 to peptide-injected animals (Fig. 5b).Representative patterns of microglial expression (Iba-1marker) are presented in Fig. 5c. Numbers of microgliawere concentrated nearby Aβ then diminished with dis-tance from deposits in both the absence (left panel) andpresence (right panel) of SB332235 treatment of rats.Overall (N = 5 animals/group), SB332235 significantlyinhibited Iba-1 ir by 57 % (Fig. 5d). The predominanceasthea33GFRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 8 of 13Fig. 5 Effects of SB332235 on area density for CXCR2 and microglia andRepresentative CXCR2 ir nearby Aβ1–42 (3 days post-Aβ1–42 injection) inscale bar is for 50 μm. b Overall CXCR2 area density (N = 5 per group) innearby Aβ1–42 in the absence (left panel) and presence (right panel) of SB(N = 5 per group) in regions within 300 μm of peptide. e Representativeof SB332235 (right panel); scale bar is for 30 μm. f Overall GFAP area densitvs Aβ1–42 + SB332235trocyte responses in proximity to peptide deposits in ML region. aabsence (left panel) and presence (right panel) of SB332235 treatment;single quadrant within 300 μm of peptide. c Representative Iba-1 ir2235 treatment; scale bar is for 30 μm. d Overall Iba-1 area densityAP ir nearby peptide deposits in the absence (left panel) and presencey (N = 5 per group) within 300 μm of peptide. *p < 0.05 for Aβ1–42of microglial staining in proximity to peptide suggestedmicroglial chemotactic responses to Aβ1–42 deposits.Typical GFAP ir in Aβ1–42-injected animals, with andwithout SB332235 treatment, is presented in Fig. 5e.Similar homogenous distributions of astrocytic ir wereevident in the presence, and absence, of CXCR2 receptorantagonist (left and right panels, Fig. 5e). Quantificationof GFAP area density (N = 5 animals/group) is presentedin Fig. 5f. The overall GFAP ir was not significantly dif-ferent between untreated peptide-injected rats or ani-mals receiving SB332235 application. In summary,microgliosis and CXCR2 area density are enhancednearby Aβ with SB332235 effective in attenuating bothresponses.Effects of SB332235 on viability of GCL neurons and lipidperoxidationNeuronal viabilityThe neuroprotective efficacy of SB332235 administrationto peptide-injected animals (3 days post-injection) wasdetermined using NeuN as a marker for GCL neurons indentate gyrus. Typical staining patterns for neurons forthe different animal groups are presented in Fig. 6a. Anintact GCL was evident in PBS control animals (upperleft panel) or animals receiving reverse peptide, Aβ42–1(not shown). Animals receiving intrahippocampal injec-tion of Aβ1–42 exhibited a marked decrease of GCL neu-rons (lower left panel). The administration of SB332235with peptide to animals markedly attenuated neuronalloss (lower right panel). Animals receiving SB332235treatment in the absence of Aβ1–42 (upper right panel)showed similar patterns of NeuN staining as for PBScontrol.Overall (N = 5 animals/group), Aβ1–42 injection causeda considerable loss of GCL neurons with levels of NeuNir diminished by 56 % compared with PBS injection(Fig. 6b). However, SB332235 treatment conferred a sig-nificant degree of neuroprotection with numbers of neu-rons increased by 36 % compared to NeuN ir withpeptide alone. Similar magnitudes of GCL neuron viabil-ity were determined for both controls (PBS and Aβ42–1)and SB332235 applied alone.Lanee ajecRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 9 of 13Fig. 6 Neuroprotective and lipid peroxidation effects of SB332235 on GC(upper left panel), SB332235 alone (upper right panel), Aβ1–42 (lower left ppost-injection; scale bar represents 50 μm. b Area density of NeuN for thperoxidation (4-HNE marker) levels following 3 days intrahippocampal in42 (lower left panel), and Aβ1–42 + SB332235 (lower right panel). Scale bar is fN = 5 per group. *p < 0.05 for Aβ1–42 vs PBS and #p < 0.05 for Aβ1–42 vs Aβ1neurons. a Representative neuronal staining (NeuN) in PBS controll), and Aβ1–42 + SB332235 (lower right panel); results are for 3 daysnimal groups, N = 5 per group. Asterisk denotes p < 0.05. c Typical lipidtion of PBS (upper left panel), SB332235 alone (upper right panel), Aβ1–or 50 μm. d Area density of 4-HNE for the different animal treatments,–42 + SB332235peptide-injected animals was effective in reducing levelsis manifest in clustering of cells in the vicinity of peptideleading to cell activation and subsequent production ofan assemblage of pro-inflammatory mediators. Our find-ings suggest microglial-derived oxidative species andlipid peroxidation could contribute to oxidative stressdamage to GCL neurons with pharmacological inhib-ition of CXCR2 efficacious in blocking inflammatory re-activity and attenuating neuronal damage.The demonstration of upregulated CXCR2 in AD vsND cortical brain tissue served as a rationale for the de-sign of animal model experiments. Importantly, corticalbrain tissue from AD individuals demonstrated areas ofCXCR2 co-localization with activated microglia. Similarresults were obtained in hippocampal brain sections inthe few cases where tissue was available. The cell-specific association of CXCR2 supports the possibilitythat microglial-mediated inflammatory responses may beinvolved in AD pathology. Involvement of CXCR2 acti-vation in inflamed brain is consistent with the findingRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 10 of 13of lipid peroxidation product (lower right panel). Thetreatment of animals with SB332235 alone was withouteffect in induction of 4-HNE (upper right panel).Quantification of data (N = 5 animals/group) indicatedlittle or no measurable 4-HNE ir in PBS or reverse pep-tide controls whereas intrahippocampal peptide injectioninduced considerable lipid peroxidation product (Fig. 6d).Antagonism of CXCR2 with SB332235 reduced levels of4-HNE (by 64 %) when applied to peptide-injected ani-mals. Application of SB332235 alone had no effect on 4-HNE area density.Effects of SB332235 on superoxideA plethora of pro-inflammatory factors have been docu-mented in AD brain [1]. Oxidative stress [34] and subse-quent neuronal degeneration in peptide-injected inflamedbrain could be mediated by superoxide production fromactivated microglia [35–37]. We used HEt as a cell-permeable probe to detect levels of superoxide adjacent tothe GCL region. No evidence for HEt ir was found inPBS-injected animal brain (upper left panel, Fig. 7a) or inanimals receiving reverse peptide injection (data notshown). Intrahippocampal Aβ1–42 injection caused amarked HEt ir (lower left panel) which was considerablyattenuated with SB332235 treatment of peptide-injectedanimals (lower right panel). SB332235 administrationalone produced minimal levels of superoxide (upper rightpanel).The extent of HEt ir is shown in bar graphs (Fig. 7b)for the different animal treatments (N = 5 animal/group).Negligible HEt staining was evident for PBS and reverseLipid peroxidationThe relevance of oxidative stress and lipid peroxidationas a contributing factor to neuronal damage and cogni-tive deficiency has been indicated [32, 33]. We investi-gated if oxidative stress might be involved in neuronaldamage in peptide-injected brain and if SB332235 couldprotect against oxidative-mediated activity. Experimentswere designed to examine the overall changes in thelipid peroxidation product 4-hydroxynonenal (4-HNE)which is produced in cells under oxidative stress. Thesemeasurements were taken in the GCL region of dentategyrus to relate oxidative effects to GCL neurons.Typical staining patterns for 4-HNE are shown inFig. 6c. Minimal 4-HNE ir was observed with PBS(upper left panel) or reverse peptide (data not shown)injections indicating a lack of oxidative damage in con-trols. However, considerable extents of 4-HNE stainingwere present in the Aβ1–42-injected rat hippocampus(lower left panel). Administration of SB332235 topeptide controls or for rats administered SB332235alone. A high HEt ir was measured in Aβ1–42-injectedrat brain with SB332235 treatment of peptide-injectedanimals significantly reducing levels of HEt ir by 55 %.DiscussionThis study presents novel findings for enhanced expres-sion of the chemokine IL-8 receptor CXCR2 in humanAD brain and in ML region of dentate gyrus in Aβ1–42-injected rat hippocampus. Evidence is presented in theAD animal model indicating upregulation of CXCR2may be linked with microglial-mediated responses whichin turn are correlated with neuronal damage in inflamedbrain. In essence, deposition of Aβ1–42 induces a micro-glial chemotactic response involving upregulation ofCXCR2 and its ligand, IL-8. A net migration of microgliaFig. 7 Effects of SB332235 on superoxide activity and inflammatoryfactors. a Representative superoxide ir (HEt) in region adjacent to GCLafter 3 days intrahippocampal injection of PBS (upper left panel),SB332235 alone (upper right panel), Aβ1–42 (lower left panel), andAβ1–42 + 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 Aβ1–42vs Aβ1–42 + SB332235that the receptor ligand, IL-8, is reported as the mosthighly upregulated factor from Aβ1–42-stimulated humanRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 11 of 13microglia [10]. Interestingly, IL-8 priming of humanmicroglia subsequently exposed to Aβ1–42 has beenfound to enhance cellular production of a host of in-flammatory factors including pro-inflammatory cyto-kines [13].The intrahippocampal injection of Aβ1–42 is an ADanimal model characterized by enhanced inflammatoryreactivity with pharmacological block of microgliosiscorrelated with increased viability of GCL neurons [25,26, 30, 38]. In the present work, expression of CXCR2and IL-8 showed similar time-dependent (1–7 days) in-creases following Aβ1–42, relative to controls (PBS andreverse peptide), intrahippocampal injection. Both re-ceptor and ligand expressions were maximal at 3 dayspost-peptide injection and remained elevated at 7 dayspost-injection. Immunohistochemical staining exhibitedsimilar results with Aβ1–42 injection yielding a fivefoldincrease in CXCR2 expression with Aβ1–42, relative toPBS, injection (time point of 3 days post-injection). Re-sults from Western blot assay showed consistent trendsin CXCR2 expression with duration of peptide injectionwith CXCR2 levels maximum at 3 days post-peptideinjection.At 3 days post-peptide injection, considerable extentsof CXCR2 immunoreactivity were co-localized withmicroglia with lesser association of receptor with astro-cytes (Fig. 3). Pharmacological antagonism of CXCR2 bySB332235 was examined with an initial focus on drug ef-fects on gliosis at 3 days subsequent to intrahippocampalinjection of Aβ1–42. A marked enhancement for bothmicrogliosis and astrogliosis was evident in ML regionof dentate gyrus compared with PBS or reverse peptideapplication (Fig. 4). Treatment of peptide-injected ani-mals with SB332235 significantly inhibited microgliosisbut was ineffective in attenuating astrogliosis. It can benoted that contributions from CXCR2-(+)ve neuronswould be minimized in sections isolated from the MLregion. In addition, the absence of myeloperoxidase(MPO) immunoreactivity (data not shown) indicatedthat CXCR2-mediated neutrophils did not contribute toinflammatory responses in Aβ1–42-injected rat brain.Microglial chemotaxis is a rapid inflammatory re-sponse to Aβ deposition in the AD model [7, 26]. In thiswork, we measured net migration of microglia in a sin-gle quadrant in the immediate vicinity of peptide de-posits in ML. Double staining was then used todetermine CXCR2 and glial ir within 300 μm of Aβ1–42deposits (Fig. 5). Animal treatment with SB332235 wasexamined for localized effects on CXCR2 area densityand microgliosis and astrogliosis. Both CXCR2 areadensity and microglial ir were significantly attenuated bySB332235 administration with no effects of the com-pound on astrocytic responses. Although this compo-nent of study does not directly target chemotacticprocesses, the results suggest efficacy for SB332235 ininhibiting microglial responses and CXCR2-dependentactivity nearby peptide.Peptide-injected (3 days) rat brain exhibited a consid-erable loss of GCL neurons compared to PBS or reversepeptide injection (Fig. 6a, b). Treatment of Aβ1–42-injected rats with SB332235 conferred a significant de-gree of neuroprotection as shown by NeuN staining inthe GCL region of dentate gyrus. Previous work usingthis animal model has demonstrated that drug actionswhich inhibit microgliosis are correlated with enhance-ment in numbers of GCL neurons [6, 26]. We also ex-amined if lipid peroxidation could contribute toneurotoxicity by assessing 4-HNE ir in the GCL region.Overall, levels of 4-HNE were markedly elevated withAβ1–42, and absent with PBS or Aβ42–1, intrahippocam-pal injection (all results obtained at 3 days post-injection). Animal treatment with SB332235 markedlyinhibited 4-HNE levels in peptide-injected brain.Previous work has demonstrated peptide-stimulatedmicroglia as a prominent source of superoxide radical [35,36]. Oxidative stress induced by superoxide species couldbe involved in the lipid peroxidation damage to neurons[34]. To examine this possibility, HEt ir was determined inthe ML region of dentate gyrus. This region was chosen tocorrespond to the areas of microglial and astrocytic re-sponses. Superoxide was not detectable in controls (PBSor Aβ42–1) at 3 days post-injection; however, considerableHEt ir was evident in Aβ1–42-injected brain. Treatment ofpeptide-injected animals with SB332235 was effective inattenuating levels of the superoxide marker. The neuro-protection conferred by SB332235 is consistent with pre-vious results showing inhibition of microgliosis as amechanism enhancing neuronal viability in the peptide-injected animal model but does not rule out possible dir-ect effects of the CXCR2 antagonist on GCL neurons.As noted above, direct intrahippocampal injection ofAβ1–42 serves as an AD animal model which exacerbatesinflammatory reactivity. The model appears to be char-acterized as one in which an acute insult evolves into achronic inflammatory perturbation in a relatively shorttime. The injection of peptide has particular utility incorrelating effects of pharmacological modulation ofmicrogliosis with viability of neurons. Validation of themodel has been considered in terms of a comparison ofcellular responses and processes with properties charac-teristic of AD brain tissue [39]. This comparison hasshown similarities in a number of features includingmicroglial and astroglial responses, abnormalities in mi-crovasculature, and leakiness in BBB. Neuronal loss ap-parent in the AD model is the correlate of cognitivedysfunction in AD brain.Our in vivo results provide evidence for efficacy ofSB332235 at a time point associated with maximal[1, 45] with diverging negative or positive effects on theRyu et al. Journal of Neuroinflammation  (2015) 12:144 Page 12 of 13viability of the neurovascular unit [46].Our findings suggest the relevance in using transgenicanimal models to examine pharmacological inhibition ofCXCR2 as a strategy to enhance cognitive function. Suchstudies would reflect the effects of a progressive buildupof peptide deposits over time, rather than direct injec-tion of amyloid, to more closely mimic chronic inflam-mation in AD brain. It should be emphasized that anumber of chemokines, their receptors, and a host ofnon-chemokine factors could contribute to inflamma-tory reactivity in the progression of AD pathology. Wesuggest the merits in using a cocktail delivery of drugsas a strategy to examine effects for modulation of mul-tiple components of chronic inflammation in treatmentof the disease.ConclusionOverall, this study has demonstrated competitive antag-onism of CXCR2 as an effective strategy in attenuatingchemokine receptor expression in microglia, the accu-mulation of microglia nearby peptide, and the cellularproduction of superoxide. The inhibition of a spectrumof inflammatory processes is correlated with an en-hanced viability of granule cell neurons. Since CXCR2and its ligand IL-8 are upregulated in AD, relative toND, brain, modulation of CXCR2 represents a novelneuroprotective strategy to be tested in other AD animalmodels.Competing interestThe authors declare that they have no competing interests.Authors’ contributionsJKR designed and conducted the research experiments and analyzed theexpression for both CXCR2 and IL-8. However, the RT-PCR data suggest that both receptor and ligand expres-sions may remain elevated over longer times. In ADbrain, expression of CXCR2 (Fig. 1) and IL-8 [12] is in-creased compared to levels in controls. In this case, theCXCR2 antagonist may have utility in reducing chronicinflammatory activity over extended times.It is important to note that beneficial effects of micro-glial response and activation have been reported in ADbrain [40–42]. Indeed, previous work on chemokine re-ceptors in Tg 2576 mice has demonstrated that attenu-ation of Ccr2 in microglia was associated with abnormalaccumulation of Aβ and increased mortality of animals[43]. Conversely, knockout of chemokine receptor Cx3cr1was found to confer neuroprotection in a mouse model ofAD [44]. Overall, a manifold of microglial-mediated in-flammatory pathways is active in peptide-stimulated brainimmunofluorescence data. TC and HBC conducted the RT-PCR and Westernblot studies and analyzed the data. NJ analyzed and interpreted theimmunofluorescence staining results from AD and ND brain tissue. JGMdesigned the overall research program, analyzed and interpreted the data,and drafted the manuscript. All authors read and approved the finalmanuscript.AcknowledgementsThe compound SB332235 was kindly provided by GlaxoSmithKline. Dr. CSchwab (UBC) and Dr. Sultan Darvish and Andrew Reid (Dalhousie University,Halifax) assisted with the analysis of AD and ND brain sections, and AldenLing and Jenny Chang assisted with the immunohistochemical staining ofhippocampal tissue from animals. This work was supported by grants fromthe Pacific Alzheimer Research Foundation and Canadian Institute of HealthResearch.Author details1Department of Anesthesiology, Pharmacology and Therapeutics, Universityof British Columbia, 2176 Health Science Mall, Vancouver, British ColumbiaV6T 1Z3, Canada. 2Brain Research Centre, University of British Columbia, 2211Wesbrook Mall, Vancouver, British Columbia, Canada. 3Department ofPharmacology, Faculty of Science, Mahidol University, Bangkok 10400,Thailand.Received: 27 January 2015 Accepted: 4 June 2015References1. Akiyama H et al. Inflammation in neurodegenerative disease. NeurobiolAging. 2000;21:383–421.2. Cameron B, Landreth GE. 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