Endogenous Prion Protein Attenuates Experimentally Induced Colitis
2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.07.025
ISSN1525-2191
AutoresGary R. Martin, Catherine M. Keenan, Keith A. Sharkey, Frank R. Jirik,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoAlthough the cellular prion protein (PrPC) is expressed in the enteric nervous system and lamina propria, its function(s) in the gut is unknown. Because PrPC may exert a cytoprotective effect in response to various physiologic stressors, we hypothesized that PrPC expression levels might modulate the severity of experimental colitis. We evaluated the course of dextran sodium sulfate (DSS)–induced colitis in hemizygous Tga20 transgenic mice (approximately sevenfold overexpression of PrPC), Prnp−/− mice, and wild-type mice. On day 7, colon length, disease severity, and histologic activity indices were determined. Unlike DSS-treated wild-type and Prnp−/− animals, PrPC overexpressing mice were resistant to colitis induction, exhibited much milder histopathologic features, and did not exhibit weight loss or colonic shortening. In keeping with these results, pro-survival molecule expression and/or phosphorylation levels were elevated in DSS-treated Tga20 mice, whereas pro-inflammatory cytokine production and pSTAT3 levels were reduced. In contrast, DSS-treated Prnp−/− mice exhibited increased BAD protein expression and a cytokine expression profile predicted to favor inflammation and differentiation. PrPC expression from both the endogenous Prnp locus or the Tga20 transgene was increased in the colons of DSS-treated mice. Considered together, these findings demonstrate that PrPC has a previously unrecognized cytoprotective and/or anti-inflammatory function within the murine colon. Although the cellular prion protein (PrPC) is expressed in the enteric nervous system and lamina propria, its function(s) in the gut is unknown. Because PrPC may exert a cytoprotective effect in response to various physiologic stressors, we hypothesized that PrPC expression levels might modulate the severity of experimental colitis. We evaluated the course of dextran sodium sulfate (DSS)–induced colitis in hemizygous Tga20 transgenic mice (approximately sevenfold overexpression of PrPC), Prnp−/− mice, and wild-type mice. On day 7, colon length, disease severity, and histologic activity indices were determined. Unlike DSS-treated wild-type and Prnp−/− animals, PrPC overexpressing mice were resistant to colitis induction, exhibited much milder histopathologic features, and did not exhibit weight loss or colonic shortening. In keeping with these results, pro-survival molecule expression and/or phosphorylation levels were elevated in DSS-treated Tga20 mice, whereas pro-inflammatory cytokine production and pSTAT3 levels were reduced. In contrast, DSS-treated Prnp−/− mice exhibited increased BAD protein expression and a cytokine expression profile predicted to favor inflammation and differentiation. PrPC expression from both the endogenous Prnp locus or the Tga20 transgene was increased in the colons of DSS-treated mice. Considered together, these findings demonstrate that PrPC has a previously unrecognized cytoprotective and/or anti-inflammatory function within the murine colon. The physiologic functions of the normal cellular prion protein (PrPC) remain poorly understood despite the well-characterized role of the misfolded molecule in the pathogenesis of neurodegenerative diseases such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Although mice lacking PrPC are viable,1Bueler H. Fischer M. Lang Y. Bluethmann H. Lipp H.P. DeArmond S.J. Prusiner S.B. Aguet M. Weissmann C. 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Absence of the cellular prion protein exacerbates and prolongs neuroinflammation in experimental autoimmune encephalomyelitis.Am J Pathol. 2008; 173: 1029-1041Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 19Hu W. Nessler S. Hemmer B. Eagar T.N. Kane L.P. Leliveld S.R. Muller-Schiffmann A. Gocke A.R. Lovett-Racke A. Ben L.H. Hussain R.Z. Breil A. Elliott J.L. Puttaparthi K. Cravens P.D. Singh M.P. Petsch B. Stitz L. Racke M.K. Korth C. Stuve O. Pharmacological prion protein silencing accelerates central nervous system autoimmune disease via T cell receptor signalling.Brain. 2010; 133: 375-388Crossref PubMed Scopus (28) Google Scholar This is of significance because these lineages produce a variety of proinflammatory cytokines that are up-regulated in the intestinal mucosa in individuals with inflammatory bowel disease.28Eastaff-Leung N. Mabarrack N. Barbour A. Cummins A. Barry S. Foxp3+ regulatory T cells: Th17 effector cells, and cytokine environment in inflammatory bowel disease.J Clin Immunol. 2010; 30: 80-89Crossref PubMed Scopus (304) Google Scholar Another possibility is that PrPC functions as an antimicrobial peptide. Pasupuleti et al29Pasupuleti M. Roupe M. Rydengard V. Surewicz K. Surewicz W.K. Chalupka A. Malmsten M. Sorensen O.E. Schmidtchen A. Antimicrobial activity of human prion protein is mediated by its N-terminal region.PLoS One. 2009; 4: e7358Crossref PubMed Scopus (65) Google Scholar have demonstrated that intact recombinant PrPC has antibacterial activity at normal and low pH and that synthetic peptides derived from the N-terminal region of PrPC are cytotoxic to several bacterial species including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Although acute colitis can accelerate the onset of disease after oral challenge with infectious prions,30Sigurdson C.J. Heikenwalder M. Manco G. Barthel M. Schwarz P. Stecher B. Krautler N.J. Hardt W.D. Seifert B. MacPherson A.J. Corthesy I. Aguzzi A. Bacterial colitis increases susceptibility to oral prion disease.J Infect Dis. 2009; 199: 243-252Crossref PubMed Scopus (29) Google Scholar a physiologic role for PrPC in colitis has not been reported. We, therefore, examined the role of PrPC in mice either lacking or overexpressing this protein in the dextran sulfate sodium (DSS)–induced model of experimental colitis. Although this model does not fully replicate human inflammatory bowel disease, it does share a number of pathologic features with this disease.31Strober W. Fuss I.J. Blumberg R.S. The immunology of mucosal models of inflammation.Annu Rev Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1145) Google Scholar, 32Wirtz S. Neufert C. Weigmann B. Neurath M.F. Chemically induced mouse models of intestinal inflammation.Nat Protoc. 2007; 2: 541-546Crossref PubMed Scopus (1150) Google Scholar Because PrPC deficiency exacerbates oxidative stress and neuroinflammation, we hypothesized that PrPC might demonstrate protective and/or anti-inflammatory effects in DSS-induced colitis. These studies were conducted in accordance with the guidelines established by the Canadian Council of Animal Care, and all protocols were approved by the Health Sciences Animal Care Committee at the University of Calgary. Male mice matched by age and body weight (20 to 25 g) were fed standard laboratory chow, had access to water ad libitum, and were maintained in a barrier unit in a room with controlled temperature (22°C ± 1°C), humidity (65% to 70%), and light cycle (12 hours light and 12 hours dark). Male hemizygous Tga20 mice (approximately sevenfold overexpression of PrPC),33Fischer M. Rulicke T. Raeber A. Sailer A. Moser M. Oesch B. Brandner S. Aguzzi A. Weissmann C. Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.EMBO J. 1996; 15: 1255-1264Crossref PubMed Scopus (780) Google Scholar Zurich I Prnp-deficient mice,1Bueler H. Fischer M. Lang Y. Bluethmann H. Lipp H.P. DeArmond S.J. Prusiner S.B. Aguet M. Weissmann C. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein.Nature. 1992; 356: 577-582Crossref PubMed Scopus (1437) Google Scholar and age-matched (8 to 10 weeks of age) wild-type (WT) littermates (all on a C57Bl/6 background) were administered 3% DSS in their drinking water for 7 days. A second group was given 3% DSS for 3 days. Mice were monitored daily to assess fluid consumption, change in body weight, stool consistency and for the presence of gross blood in the feces. Mice were euthanized at day 3 or 7 after initiation of treatment using DSS or tap water, and their colons were quickly removed. The severity of colitis was assessed using an established Disease Activity Index (DAI) demonstrated to be relatively representative of clinical measures for assessment of inflammatory bowel disease.34Cooper H.S. Murthy S.N. Shah R.S. Sedergran D.J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar The DAI score was derived as follows: for weight loss, 0 = none, 1 = 1% to 5%, 2 = 5% to 10%, 3 = 10% to 15%, and 4 = greater than 15%; for stool consistency, 0 = normal, 2 = loose, and 4 = watery diarrhea; and for colonic bleeding, 0 = none, 2 = slight, and 4 = gross. The scores were added to produce a DAI ranging from 0 (unaffected) to 12 (severe colitis). Colonic samples (3 to 4 cm distal to the cecum) were fixed in 10% neutral buffered formalin, embedded in paraffin, and stained using either H&E or periodic acid–Schiff. Damage was assessed using a previously described histologic activity index (HAI),34Cooper H.S. Murthy S.N. Shah R.S. Sedergran D.J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar as follows: for epithelial damage, 0 = none, 1 = minimal loss of goblet cells, 2 = extensive loss of goblet cells, 3 = loss of crypt cells and extensive loss of goblet cells, and 4 = extensive loss of crypt cells and goblet cells; and for infiltration, 0 = none, 1 = crypt base, 2 = muscularis mucosa, 3 = extensive muscularis mucosa and edema, and 4 = submucosal. Intestinal permeability was determined in healthy untreated mice by measuring levels of plasma fluorescein isothiocyanate (FITC)–labeled dextran35Kiela P.R. Laubitz D. Larmonier C.B. Midura-Kiela M.T. Lipko M.A. Janikashvili N. Bai A. Thurston R. Ghishan F.K. Changes in mucosal homeostasis predispose NHE3 knockout mice to increased susceptibility to DSS-induced epithelial injury.Gastroenterology. 2009; 137 (975 e961-e910): 965-975Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar after administration via gavage of 60 mg/100 g body weight of FITC-conjugated dextran (average molecular mass 4000 Da; Sigma-Aldrich Corp., St. Louis, MO) in PBS (pH 7.4). Blood was obtained by cardiac puncture 4 hours after administration of FITC-conjugated dextran and centrifuged for 10 minutes (5000 rpm at 4°C). The plasma (100 μL) was added to a 96-well dark microplate, and the concentration of fluorescein was determined using spectrophotofluorometry (Fluoroskan Ascent; Thermo LabSystems, Inc., Beverly, MA) with an excitation wavelength of 485 nm and an emission wavelength of 527 nm, using serially diluted samples of the marker as standard. To determine PrPC expression, colonic homogenates (10% w/v) were prepared in extraction buffer (0.15 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton-X 100, 10 mmol/L Tris-HCl [pH 7.4]) with the addition of a protease inhibitor cocktail (Complete; Roche Diagnostics GmbH, Mannheim, Germany). Protein concentrations were determined using the Bradford assay. Proteins (30 μg) were separated on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Lysates were prepared from mouse brain for use as a quality control measure. Membranes were blocked in 5% skim milk–TBST (Tris-buffered saline and Tween 20) and incubated with anti-PrP monoclonal antibodies SAF-32 (1:4000 dilution; Cayman Chemical Co., Inc., Ann Arbor, MI) or BAR-221 (1:4000 dilution; SPIbio, BioNovus Life Sciences, Cherrybrook, NSW, Australia) for 24 hours at 4°C. For determination of PrPC expression in the colon, the primary antibody used was SAF-32. A horseradish peroxidase–conjugated sheep anti-mouse secondary antibody was used. Bands were visualized using a chemiluminescence substrate (SuperSignal West Pico; Thermo Scientific Pierce Protein Research Products, Rockford, IL), and densities were assessed using a calibrated imaging densitometer equipped with Quantity One software (Bio-Rad, Mississauga, ON, Canada). Filters were then stripped and re-probed using an anti–β-actin antibody (Sigma-Aldrich Corp.). To determine pro-survival and pro-apoptotic signaling molecule expression, methods were duplicated as described above with modifications. Proteins (50 μg) from each sample were separated on 10% SDS-polyacrylamide gels, and after transfer, membranes were blocked in 5% skim milk–TBST, washed three times in TBST, and incubated overnight at 4°C in 5% bovine serum albumin with the polyclonal antibodies p44/42 (1:1000), phospho-p44/42 (1:500), phospho-Akt (1:500), phospho-STAT3 (1:500), p38 (1:1000), and phospho-p38 (all from Cell Signaling Technology, Inc., Beverly, MA). The mouse colons (3 to 4 cm distal to the cecum) were removed, opened along the midline, and placed in PBS containing 1 μmol/L nifedipine to fully relax the smooth muscle. After 5 minutes, these segments were pinned flat, fixed in 10% formalin for 48 hours, embedded in paraffin, and sectioned (6 μmol/L). After deparaffinization in xylene and rehydration in graded ethanol, endogenous peroxidase activity was blocked via incubation with 3% hydrogen peroxide. Tissue sections were then heated at 100°C in 10 mmol/L citrate buffer (pH 6.0) to retrieve antigens, and were preincubated with normal goat serum (10%). The primary antibody used for PrPC staining was mouse monoclonal anti–SAF-32 (1:500; overnight incubation at 4°C; Cayman Chemical Co., Inc.), and the secondary antibody used was donkey anti-mouse CY3 antibody (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The tissues were subsequently mounted and visualized using an AxioImager A2 fluorescence microscope in conjunction with an AxioCam MR3 digital monochrome camera and AxioVision 4 LE software (all from Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Micrographs (40× magnification) were obtained under identical conditions. Real-time RT-PCR was used to examine transcriptional changes of several cytokines, as well as regulators of cytokine expression that have demonstrated a role in colitis. These genes included Il1b, Il6, Il10, Il17A, Tgfb, Tnfa, Ifng, Socs3 and Nos2. Colonic tissues from control and DSS-treated mice were placed in TRIzol reagent (Gibco-BRL, Invitrogen Corp., Carlsbad, CA), and total cellular RNA was extracted according to the manufacturer's instructions. First-strand cDNA was synthesized from 2 μg total RNA in a 25-μL reaction volume. Duplicate independent quantitative real-time PCR was performed using the LightCycler System (Roche Diagnostics GmbH). SYBR Green I (Roche Diagnostics GmbH) was used to visualize and monitor the amplified product in real time. Gene-specific oligonucleotide primers were designed for the genes of interest. β-Actin was amplified as an internal control. The change in fluorescence of SYBR Green I dye in each cycle was determined using the LightCycler system software (Roche Diagnostics GmbH), and the threshold cycle above background for each reaction was calculated. The Ct value of β-actin was subtracted from that of the gene of interest to obtain a Ct value. The Ct value of an arbitrary calibrator (eg, WT control group) was subtracted from the Ct value of each sample to obtain a Ct value. The transcription level relative to the calibrator was expressed as 2−Ct. Primers used were β-actin (forward, 5′-AGAGGGAAATCGTGCGTGAC-3′; reverse, 5′-CAATAGTGATGACCTGGCCGT-3′), IFN-γ (forward, 5′-TCAAGTGGCATAGATGTGGAAGAA-3′; reverse, 5′-TGGCTCTGCAGGATTTTCATG-3′), iNOS (forward, 5′-CAGCTGGGCTGTACAAACCTT-3′; reverse, 5′-CATTGGAAGTGAAGCGTTTCG-3′), IL-1β (forward, 5′-CAACCAACAAGTGATATTCTCCATG-3′; reverse, 5′-GATCCACACTCTCCAGCTGCA-3′), IL-6 (forward, 5′-TCCAGTTGCCTTCTTGGGAC-3′; reverse, 5′-GTGTAATTAAGCCTCCGACTTG-3′), IL-10 (forward, 5′-GGTTGCCAAGCCTTATCGGA-3′; reverse, 5′-ACCTGCTCCACTGCCTTGCT-3′), IL-17 (forward, 5′-GAAGCTCAGTGCCGCCA-3′; reverse, 5′-TTCATGTGGTGGTCCAGCTTT-3′), SOCS3 (forward, 5′-GGGTGGCAAAGAAAAGGAG-3′; reverse, 5′-GTTGAGCGTCAAGACCCAGT-3′), TGF-β (forward, 5′-TGACGTCACTGGAGTTGTACGG-3′; reverse, 5′-GGTTCATGTCATGGATGGTGC-3′), and TNF-α (forward, 5′-CATCTTCTCAAAATTCGAGTGACAA-3′; reverse, 5′-TGGGAGTAGACAAGGTACAAC CC-3′). Results are expressed as mean ± SE. Comparisons among groups of data were made using one-way analysis of variance followed by a post hoc Tukey's test. P < 0.05 was considered significant. Treatment with DSS significantly reduced body weight in all groups when compared with untreated mice (Figure 1A). However, weight loss was decreased at 5 to 7 days after initiation of treatment in PrPC overexpressing mice when compared with either Prnp−/− or WT mice. These overexpressing mice also demonstrated attenuation of colonic shortening when compared with Prnp−/− or WT mice (Figure 1B). Colonic morphologic damage induced by DSS was markedly reduced in mice that overexpressed PrPC (Figure 2, A–D). DSS treatment increased colonic damage in all groups when compared with control mice given tap water, however, DAIs from Tga20 mice were significantly reduced relative to Prnp−/− or WT mice (Figure 2E). Although there were elevations in HAIs in all mice receiving DSS, the degree of damage in mice that overexpressed PrPC was decreased relative to WT or Prnp−/− mice (Figure 2F). In healthy colonic tissue, DAI and HAI values for Prnp−/− and Tga20 mice were identical to those for healthy WT mice (data not shown). No structural colonic abnormalities were observed among the various groups of healthy mice (Figure 3). However, there were marked reductions in DSS-induced colonic epithelial damage and crypt cell loss in Tga20 mice relative to similarly treated WT and Prnp−/− mice (Figure 3). Moreover, leukocyte infiltration, muscle thickening, and edema were markedly reduced in Tga20 mice when compared with either Prnp−/− or WT mice. Periodic acid–Schiff staining revealed that brush border membrane and goblets cell losses were attenuated in the colonic tissues of DSS-treated Tga20 mice when compared with similarly treated WT and Prnp−/− mice. These losses of brush border membrane and goblet cells seemed to be exacerbated in Prnp-deficient mice.Figure 3Histologic damage is reduced in colonic tissues of DSS-treated Tga20 mice. H&E staining was used to visualize colonic samples from healthy and DSS-treated mice. No histologic abnormalities were observed in untreated mice (left panels). With DSS treatment, epithelial damage, depicted by loss of goblet cells, crypt cells, and brush border, was exacerbated in WT and Prnp−/− mice as compared with Tga20 mice. In addition, infiltration was reduced in Tga20 mice as compared with the other groups. Periodic acid–Schiff staining of sections from DSS-treated mice revealed preservation of colonic brush border (black arrowheads) and goblet cells (yellow arrowheads) in Tga20 mice as compared with WT and Prnp−/− mice. Scale bar = 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunoblotting was performed to determine the levels of PrPC expression in the colon of WT, Prnp−/−, and Tga20 mice, both before and after induction of colitis. Initially, PrPC expression was verified using brain lysates as a positive control and immunoblotting with two different antibodies: SAF-32, which recognizes an epitope within the octarepeat regio
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