Adiponectin Deficiency Protects Mice From Chemically Induced Colonic Inflammation
2006; Elsevier BV; Volume: 132; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2006.11.026
ISSN1528-0012
AutoresRaja Fayad, Maria Pini, Joseph A. Sennello, Robert J. Cabay, Lawrence Chan, Aimin Xu, Giamila Fantuzzi,
Tópico(s)Biomarkers in Disease Mechanisms
ResumoBackground & Aims: Adiponectin (APN) is an adipokine that regulates insulin sensitivity and is anti-inflammatory in atherosclerosis. The goal of this study was to investigate the role of APN in intestinal inflammation. Methods: APN knockout (KO) mice and their wild-type (WT) littermates received dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce intestinal inflammation. Clinical and histologic scores and proliferation of epithelial cells were assessed. Cytokines and APN levels were measured. Expression of APN and heparin binding epidermal growth factor (HB-EGF) was analyzed by immunohistochemistry. Expression of APN and its receptors, HB-EGF, and basic fibroblast growth factor (bFGF) messenger RNA was assessed by reverse-transcription polymerase chain reaction. Association of serum APN with HB-EGF and bFGF was studied by coimmunoprecipitation. Results: APN KO mice are protected from chemically induced colitis; administration of APN restores inflammation. APN is expressed in the colon, luminal APN associates with colonic epithelial cells. In vitro, APN increases production of proinflammatory cytokines from colonic tissue. Expression of colonic APN overlaps with that of bFGF and HB-EGF, which play a protective role in colitis. Circulating APN binds to bFGF and HB-EGF, likely inhibiting their protective activity. Inhibition of EGF receptor signaling, which is required for biologic activity of HB-EGF, restores inflammation in APN KO mice. Conclusions: APN deficiency is associated with protection from chemically induced colitis. APN exerts proinflammatory activities in the colon by inducing production of proinflammatory cytokines and inhibiting bioactivity of protective growth factors. Thus, in colitis, APN exerts an opposite role compared with atherosclerosis. Background & Aims: Adiponectin (APN) is an adipokine that regulates insulin sensitivity and is anti-inflammatory in atherosclerosis. The goal of this study was to investigate the role of APN in intestinal inflammation. Methods: APN knockout (KO) mice and their wild-type (WT) littermates received dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce intestinal inflammation. Clinical and histologic scores and proliferation of epithelial cells were assessed. Cytokines and APN levels were measured. Expression of APN and heparin binding epidermal growth factor (HB-EGF) was analyzed by immunohistochemistry. Expression of APN and its receptors, HB-EGF, and basic fibroblast growth factor (bFGF) messenger RNA was assessed by reverse-transcription polymerase chain reaction. Association of serum APN with HB-EGF and bFGF was studied by coimmunoprecipitation. Results: APN KO mice are protected from chemically induced colitis; administration of APN restores inflammation. APN is expressed in the colon, luminal APN associates with colonic epithelial cells. In vitro, APN increases production of proinflammatory cytokines from colonic tissue. Expression of colonic APN overlaps with that of bFGF and HB-EGF, which play a protective role in colitis. Circulating APN binds to bFGF and HB-EGF, likely inhibiting their protective activity. Inhibition of EGF receptor signaling, which is required for biologic activity of HB-EGF, restores inflammation in APN KO mice. Conclusions: APN deficiency is associated with protection from chemically induced colitis. APN exerts proinflammatory activities in the colon by inducing production of proinflammatory cytokines and inhibiting bioactivity of protective growth factors. Thus, in colitis, APN exerts an opposite role compared with atherosclerosis. Adiponectin (APN) is a protein produced and secreted mainly by adipocytes and is thus considered a member of the growing family of adipokines.1Kim J.Y. Scherer P.E. Adiponectin, an adipocyte-derived hepatic insulin sensitizer regulation during development.Pediatr Endocrinol Rev. 2004; 1: 428-431PubMed Google Scholar APN is an abundant protein in the circulation, being present in blood of healthy individuals at concentrations of approximately 5–20 μg/mL.1Kim J.Y. Scherer P.E. Adiponectin, an adipocyte-derived hepatic insulin sensitizer regulation during development.Pediatr Endocrinol Rev. 2004; 1: 428-431PubMed Google Scholar This adipokine shares structural homologies with complement factor C1q and with the cytokine tumor necrosis factor α (TNF-α).1Kim J.Y. Scherer P.E. Adiponectin, an adipocyte-derived hepatic insulin sensitizer regulation during development.Pediatr Endocrinol Rev. 2004; 1: 428-431PubMed Google Scholar APN has a complex structure. The monomer is composed of a collagenous and a globular domain: APN monomers assemble into trimers, which further polymerize to various degrees, leading to the presence of several molecular weight forms of APN in the circulation.2Waki H. Yamauchi T. Kamon J. Ito Y. Uchida S. Kita S. Hara K. Hada Y. Vasseur F. Froguel P. Kimura S. Nagai R. Kadowaki T. Impaired multimerization of human adiponectin mutants associated with diabetes Molecular structure and multimer formation of adiponectin.J Biol Chem. 2003; 278: 40352-40363Crossref PubMed Scopus (877) Google Scholar Furthermore, APN can be cleaved by elastase, resulting in the generation of a free globular domain, which may have distinct biologic functions.3Waki H. Yamauchi T. Kamon J. Kita S. Ito Y. Hada Y. Uchida S. Tsuchida A. Takekawa S. Kadowaki T. Generation of globular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1.Endocrinology. 2005; 146: 790-796Crossref PubMed Scopus (275) Google Scholar APN exerts its activity using 2 major mechanisms. The first mechanism involves binding to specific 7-transmembrane domain receptors, which have been cloned and named ADIPOR1 and ADIPOR2.4Kadowaki T. Yamauchi T. Adiponectin and adiponectin receptors.Endocr Rev. 2005; 26: 439-451Crossref PubMed Scopus (2166) Google Scholar These receptors are expressed by a variety of cell types and signal through multiple pathways, including adenosine monophosphate (AMP) kinase and p38 mitogen-activated protein (MAP) kinase.4Kadowaki T. Yamauchi T. Adiponectin and adiponectin receptors.Endocr Rev. 2005; 26: 439-451Crossref PubMed Scopus (2166) Google Scholar In addition to binding to its own receptors, APN can also act as a decoy for several growth factors, including basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and heparin binding epidermal growth factor (HB-EGF).5Wang Y. Lam K.S. Xu J.Y. Lu G. Xu L.Y. Cooper G.J. Xu A. Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerization-dependent manner.J Biol Chem. 2005; 280: 18341-18347Crossref PubMed Scopus (334) Google Scholar In this manner, APN impedes growth factors from activating their respective receptors and therefore effectively prevents their bioactivity. Furthermore, APN binds to T cadherin, although the biologic significance of this interaction is still unclear.6Hug C. Wang J. Ahmad N.S. Bogan J.S. Tsao T.S. Lodish H.F. T-cadherin is a receptor for hexameric and high-molecular-weight forms of Acrp30/adiponectin.Proc Natl Acad Sci U S A. 2004; 101: 10308-10313Crossref PubMed Scopus (716) Google Scholar The biology of APN has mostly been investigated in the context of insulin sensitivity and atherosclerosis. A consistent epidemiologic relationship between low circulating APN and diabetes, metabolic syndrome, and cardiovascular disease has been reported by several groups.1Kim J.Y. Scherer P.E. Adiponectin, an adipocyte-derived hepatic insulin sensitizer regulation during development.Pediatr Endocrinol Rev. 2004; 1: 428-431PubMed Google Scholar Whereas the effect of APN in regulating insulin sensitivity appears to be mainly mediated by binding to ADIPOR1 and/or 2, the role of this adipokine in atherosclerosis is at least partly due to its decoy activity toward molecules that act as growth factors for smooth muscle cells, such as PDGF and HB-EGF.7Shimada K. Miyazaki T. Daida H. Adiponectin and atherosclerotic disease.Clin Chim Acta. 2004; 344: 1-12Crossref PubMed Scopus (207) Google Scholar Furthermore, several anti-inflammatory effects have been described for APN, including inhibition of TNF-α production and activity, inhibition of nuclear factor (NF)-κB activation, and induction of anti-inflammatory cytokines, as well as down-regulation of adhesion molecules.8Fantuzzi G. Adipose tissue, adipokines, and inflammation.J Allergy Clin Immunol. 2005; 115: 911-920Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar Based on these observations and on the protective role of APN in cardiovascular disease, this adipokine is generally considered as an anti-inflammatory molecule. However, more recently, this concept has been put into question by reports indicating that APN may also exert proinflammatory effects. In particular, a proinflammatory role for APN has been suggested in the context of arthritis by the demonstration that APN selectively induces production of interleukin (IL-) 6 and matrix metalloproteinase-1 from synovial fibroblasts.9Ehling A. Schaffler A. Herfarth H. Tarner I.H. Anders S. Distler O. Paul G. Distler J. Gay S. Scholmerich J. Neumann E. Muller-Ladner U. The potential of adiponectin in driving arthritis.J Immunol. 2006; 176: 4468-4478PubMed Google Scholar Furthermore, in the human colonic epithelial cell line HT-29, APN stimulates proliferation and production of proinflammatory cytokines, with a potentially distinct role of the globular fragment compared with full-length APN.10Ogunwobi O.O. Beales I.L. Adiponectin stimulates proliferation and cytokine secretion in colonic epithelial cells.Regul Pept. 2006; 134: 105-113Crossref PubMed Scopus (129) Google Scholar Thus, the pro- vs anti-inflammatory activities of APN may be cell- and/or context dependent. Despite these interesting in vitro observations on the potential proinflammatory effect on APN in the colon, in vivo data have yet to be reported. The possible role of APN in regulating inflammation in the colon is underscored by observations indicating the presence of increased APN production in inflamed mesenteric adipose tissue (fat wrapping) in patients with Crohn's disease, as well as increased circulating APN levels in patients with both ulcerative colitis and Crohn's disease.11Yamamoto K. Kiyohara T. Murayama Y. Kihara S. Okamoto Y. Funahashi T. Ito T. Nezu R. Tsutsui S. Miyagawa J.I. Tamura S. Matsuzawa Y. Shimomura I. Shinomura Y. Production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in Crohn's disease.Gut. 2005; 54: 789-796Crossref PubMed Scopus (220) Google Scholar, 12Karmiris K. Koutroubakis I.E. Xidakis C. Polychronaki M. Voudouri T. Kouroumalis E.A. Circulating levels of leptin, adiponectin, resistin, and ghrelin in inflammatory bowel disease.Inflamm Bowel Dis. 2006; 12: 100-105Crossref PubMed Scopus (283) Google Scholar In this report, we investigated the role of APN in regulating intestinal inflammation using chemically induced models of inflammatory colitis in mice. Because APN appears to be a critical factor in regulating cell proliferation as well as inflammation in atherosclerosis,7Shimada K. Miyazaki T. Daida H. Adiponectin and atherosclerotic disease.Clin Chim Acta. 2004; 344: 1-12Crossref PubMed Scopus (207) Google Scholar the models of dextran sulfate sodium (DSS)- and trinitrobenzene sulfonic acid (TNBS)-induced colitis were utilized to study the role of APN in colitis. In fact, disruption of epithelial cell proliferation, as well as production of proinflammatory mediators in the colonic mucosa are the 2 major mechanisms mediating disease in response to administration of DSS or TNBS. We demonstrate that APN knockout (KO) mice are protected from DSS- and TNBS-induced colitis and suggest that the protective effect of APN deficiency is mediated by both the decoy activity of APN toward growth factors that help maintain and repair integrity of colonic epithelium and by a proinflammatory role of APN on colonic mucosa. Our findings are in disagreement with data reported by Nishihara et al13Nishihara T. Matsuda M. Araki H. Oshima K. Kihara S. Funahashi T. Shimomura I. Effect of adiponectin on murine colitis induced by dextran sulfate sodium.Gastroenterology. 2006; 131: 853-861Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar while this paper was under review. Possible reasons for the opposite findings obtained by the 2 groups are presented in the Discussion section. Eight- to 10-week-old female APN KO mice and their +/− or +/+ littermates in a C57BL6 background were generated as previously described.14Ma K. Cabrero A. Saha P.K. Kojima H. Li L. Chang B.H. Paul A. Chan L. Increased β oxidation but no insulin resistance or glucose intolerance in mice lacking adiponectin.J Biol Chem. 2002; 277: 34658-34661Crossref PubMed Scopus (283) Google Scholar No significant differences in serum APN levels were observed between +/− and +/+ mice, and therefore both genotypes were used as wild-type (WT) controls. This strain of KO mice does not develop insulin resistance or other major metabolic abnormalities.14Ma K. Cabrero A. Saha P.K. Kojima H. Li L. Chang B.H. Paul A. Chan L. Increased β oxidation but no insulin resistance or glucose intolerance in mice lacking adiponectin.J Biol Chem. 2002; 277: 34658-34661Crossref PubMed Scopus (283) Google Scholar Furthermore, analysis of immune cell subpopulations in blood, spleen, and thymus, as well as histologic analysis of colonic tissue did not reveal any significant difference compared with WT littermates (data not shown). Care of mice followed institutional guidelines under a protocol approved by the Institutional Animal Care and Use Committee at the University of Illinois at Chicago. To induce colonic inflammation, WT and KO mice (5 to 7 mice per group) received 2% DSS (molecular mass, 40 kilodaltons; MP Biochemicals, Solon, OH) dissolved in drinking water for experimental days 1–5, followed by 5 days of regular drinking water. On day 5 or 10, mice were killed. Control mice had access to drinking water without DSS. A group of KO mice (KO-APN) were injected daily with 30 μg of recombinant murine APN (Alexis Biochemicals, San Diego, CA) intraperitoneally (IP) until killed. Recombinant APN was produced in mammalian HEK-293 cells; the molecule forms both hexamers and high molecular weight (MW) complexes, thus mimicking the structure of adipocyte-derived APN (Table 3). Bioactivity of the same preparation of recombinant APN was previously demonstrated by its ability to reduce serum alanine transferase levels in lipodystrophic mice.15Sennello J.A. Fayad R. Morris A.M. Eckel R.H. Asilmaz E. Montez J. Friedman J.M. Dinarello C.A. Fantuzzi G. Regulation of T-cell-mediated hepatic inflammation by adiponectin and leptin.Endocrinology. 2005; 146: 2157-2164Crossref PubMed Scopus (78) Google Scholar Individual mice were observed daily for clinical signs of disease that are manifested by weight loss, positive fecal hemoccult, and diarrhea. The clinical score was assessed in a blinded fashion, as previously described.16Siegmund B. Rieder F. Albrich S. Wolf K. Bidlingmaier C. Firestein G.S. Boyle D. Lehr H.A. Loher F. Hartmann G. Endres S. Eigler A. Adenosine kinase inhibitor GP515 improves experimental colitis in mice.J Pharmacol Exp Ther. 2001; 296: 99-105PubMed Google Scholar For TNBS experiments, mice received 2 mg TNBS in 50% ethanol intrarectally on days 1 and 7 and were killed on day 16. Control mice received 50% ethanol without TNBS as vehicle. Parameters evaluated were the same as reported for the DSS model.Table 3Percentage Distribution of Recombinant APN as well as APN Present in Serum or Colon Culture Supernatant Into High and Middle/Low Molecular Weight ComplexesMolecular weight formSerumColon culture supernatant from DSS-treated WT (%)Murine recombinant APN (%)WT (%)APN-injected KO (%)HMW62.1958.9460.8858.88MMW/LMW37.8141.0639.1241.12NOTE. Data are expressed as percentage of HMW or MMW/LMW APN after fractionation as described in the Materials and Methods section. Open table in a new tab NOTE. Data are expressed as percentage of HMW or MMW/LMW APN after fractionation as described in the Materials and Methods section. To evaluate DSS- or TNBS-induced colitis histologically, the entire colon was excised, and a 0.5- to 1-cm segment of the transverse colon was fixed in 10% buffered formalin overnight for histologic analysis. Paraffin sections (5 μm) were stained with H&E. Histologic scoring was performed in a blinded fashion by a pathologist as previously described.16Siegmund B. Rieder F. Albrich S. Wolf K. Bidlingmaier C. Firestein G.S. Boyle D. Lehr H.A. Loher F. Hartmann G. Endres S. Eigler A. Adenosine kinase inhibitor GP515 improves experimental colitis in mice.J Pharmacol Exp Ther. 2001; 296: 99-105PubMed Google Scholar, 17Atreya R. Mudter J. Finotto S. Mullberg J. Jostock T. Wirtz S. Schutz M. Bartsch B. Holtmann M. Becker C. Strand D. Czaja J. Schlaak J.F. Lehr H.A. Autschbach F. Schurmann G. Nishimoto N. Yoshizaki K. Ito H. Kishimoto T. Galle P.R. Rose-John S. Neurath M.F. Blockade of interleukin 6 transsignaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in Crohn's disease and experimental colitis in vivo.Nat Med. 2000; 6: 583-588Crossref PubMed Scopus (1102) Google Scholar To detect local APN and HB-EGF expression, a 0.5- to 1-cm segment of the transverse colon was fixed in 10% buffered formalin overnight, and paraffin sections (5 μm) were stained for APN or HB-EGF using goat antimurine APN (R&D Systems, Minneapolis, MN) or goat antimurine HB-EGF (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies, and the reaction was developed using diaminobenzidine (DAB). We performed 2 sets of controls to ensure specificity of the staining: (1) sections were stained with goat immunoglobulin (Ig) G; (2) antibodies were preincubated with the respective recombinant proteins (10 μg/mL) for 1 hour at room temperature before performing the staining procedure. For evaluation of epithelial cell proliferation, each mouse received 1 mg bromodeoxyuridine (BrdU) (Sigma Chemical Co, St. Louis, MO) IP 2 hours before death. A 0.5- to 1-cm segment of the transverse colon was fixed in 10% buffered formalin overnight and embedded in paraffin, and tissue sections (5 μm) were stained for BrdU using a BrdU-staining kit from BD Biosciences (San Jose, CA). The labeling index (number of BrdU-positive cells per 2 mm2 of mucosa) was determined using a Zeiss microscope with a video camera and a monitor. An average of 2 mm2 of mucosa was defined in each sample to count BrdU-positive cells, and at least 15 well-defined crypts were counted from each animal. Two millimeters2 of colon mucosa was used as the area of BrdU-positive cells in animals with severe colitis and total destruction of colon crypts. All cell counts were carried out independently by an observer blinded to the study design. A segment of the transverse colon was removed, cut open longitudinally, and washed in phosphate-buffered saline (PBS) containing penicillin and streptomycin. Colon cultures were performed as previously described, in the absence or presence of recombinant APN at 10 μg/mL.18Siegmund B. Sennello J.A. Jones-Carson J. Gamboni-Robertson F. Lehr H.A. Batra A. Fedke I. Zeitz M. Fantuzzi G. Leptin receptor expression on T lymphocytes modulates chronic intestinal inflammation in mice.Gut. 2004; 53: 965-972Crossref PubMed Scopus (130) Google Scholar Culture supernatants were harvested and assayed for IL-6, macrophage inflammatory protein (MIP) 2, bFGF, and APN levels using enzyme-linked immunosorbent assays (ELISA) kits from BD Bioscience and R&D Systems. Protein concentration was determined using a Bio-Rad (Hercules, CA) protein assay. Concentrations of the different mediators were adjusted by total protein content. Total RNA was extracted from colonic and adipose tissues and reverse transcribed prior to performing polymerase chain reaction (PCR) (30 cycles) using the following primers: APN forward: 5′-GACGTTACTACAACTGAAGAGC-3′, APN reverse: 5′-CATTCTTTTCCTGATACTGGTC-3′; ADIPOR1 forward: 5′-ACGTTGGAGAGTCATCCCGTAT-3′, ADIPOR1 reverse: 5′-AGTGTGGAAGAGCCAGGAGA-3′; ADIPOR2 forward: 5′-TCCCAGGAAGATGAAGGGTTTAT- 3′, ADIPOR2 reverse: 5′-CCATGAAAAGGAAAGGCAGA-3′; bFGF forward: 5′-AGCGACCCACACGTCAAACT-3′, bFGF reverse: 5′-CGTCCATCTTCCTTCATAGCAAG-3′; HB-EGF forward: 5′-TTTGGAGAGTCCTTTGCAGA-3′, HB-EGF reverse: 5′-TGTGACAATGAGATTCCTTGTG-3′; GAPDH forward: 5′-ACCACAGTCCATGCCATCAC-3′, GAPDH reverse: 5′-TCCACCACCCTGTTGCTGTA-3′. Quantitative real-time reverse-transcription (RT)-PCR for APN was performed as previously described.19Chen B. Lam K.S. Wang Y. Wu D. Lam M.C. Shen J. Wong L. Hoo R.L. Zhang J. Xu A. Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes.Biochem Biophys Res Commun. 2006; 341: 549-556Crossref PubMed Scopus (178) Google Scholar Expression of APN was normalized by calibrating against expression of 18S RNA. WT mice and KO (7 per group) received 2% DSS in drinking water for 5 days concurrently with 1 mg/mouse of Tyrphostin (Tyr) (AG 1748, LC Laboratories, Woburn, MA) dissolved in 0.1% of dimethylsulfoxide (DMSO) in PBS. Tyr was injected IP once daily until death on day 10. Control mice were injected with DMSO/PBS as vehicle using the same time schedule. Dose and scheduling of Tyr administration were determined based on previously published reports.20Johns T.G. Luwor R.B. Murone C. Walker F. Weinstock J. Vitali A.A. Perera R.M. Jungbluth A.A. Stockert E. Old L.J. Nice E.C. Burgess A.W. Scott A.M. Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478.Proc Natl Acad Sci U S A. 2003; 100: 15871-15876Crossref PubMed Scopus (84) Google Scholar Albumin and immunoglobulin G from C57BL/6 mouse serum (∼10 mL) were depleted by the ProteoExtract Albumin/IgG Removal Kit (Calbiochem, Merck Biosciences, Darmstadt, Germany), and the remaining supernatant was incubated with 200 μL Sepharose beads coupled with rabbit nonimmune IgG to remove nonspecific bindings. The clarified supernatant was then incubated with 100 μL Sepharose beads coupled with anti-mouse adiponectin IgG at 4°C overnight. The beads were washed with TBS and the bound protein complexes eluted with 0.1 mol/L glycine-HCl (pH 2.5). The eluted fractions were concentrated, separated by 15% SDS-PAGE, and visualized by Coomassie brilliant blue (CBB) staining. To detect FGF or HB-EGF, Western blot analysis of the concentrated samples using antibasic FGF antibody (Upstate, Charlottesville, VA) or anti-HB-EGF (R&D system) was performed, respectively. Murine recombinant APN (100 ng) serum obtained from WT mice or APN-injected KO mice (10 μL) or supernatant of colon cultures obtained from DSS-treated WT mice (300 μL) were fractionated as previously described.21Morris A.M. Sennello J.A. Fayad R. Eckel R.H. Dinarello C.A. Fantuzzi G. T-cell-mediated hepatic inflammation modulates adiponectin levels in mice: role of tumor necrosis factor α.Metabolism. 2006; 55: 555-559Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Data are expressed as mean ± SEM. The statistical significance of differences between treatment and control groups was determined by factorial ANOVA. Statistical analyses were performed using the XLStat software (Addinsoft, Brooklyn, NY). To study the role of APN in intestinal inflammation, we compared the response to DSS in WT and APN KO mice. Starting from day 3 of DSS administration, WT mice showed signs of intestinal inflammation, manifested by weight loss, diarrhea, and positive fecal hemoccult (combined in a clinical score in Figure 1A). In contrast, DSS-induced inflammation was delayed and significantly reduced in KO mice (Figure 1A). Administration of recombinant APN to DSS-treated KO mice (KO-APN), which restored systemic APN levels to ∼50% of those present in WT mice, reconstituted the inflammatory response to levels comparable with those observed in WT mice. As shown in Figure 1C and quantified in Figure 1B, the colon of WT mice receiving DSS was characterized by an inflammatory infiltrate and destruction of epithelial cell architecture at both days 5 and 10. In stark contrast, KO mice treated with DSS did not show inflammation or disruption of crypt architecture at either time point. Reconstitution with exogenous APN restored the colonic inflammatory response of DSS-treated KO mice The lack of inflammation in the colon of DSS-treated KO mice and the ability of exogenous APN to restore inflammation were also confirmed by measurement of the proinflammatory cytokines IL-6 and MIP2 in colon culture supernatants (Figure 1D). Proliferation of epithelial cells is necessary to maintain and restore epithelial integrity during and following damage induced by DSS. A dramatic reduction in epithelial cell proliferation was observed in DSS-treated WT mice as well as in DSS-treated KO mice injected with exogenous APN. In contrast, DSS did not significantly alter epithelial cell proliferation in KO mice (Figure 1C and Table 1).Table 1Quantification of Colonic Epithelial Cell Proliferation in WT and KO Mice Receiving DSSControlaData are mean ± SEM of the number of BrdU-positive epithelial cells per 2 mm2 of colonic mucosa. N = 3 mice per group.5 days10 daysWT58.3 ± 9.26.6 ± 3.3bP < .01 vs WT control.12.6 ± 2.8bP < .01 vs WT control.KO87.3 ± 8.960.0 ± 6.6cP < .01 vs WT treated with DSS at the same time point.118.3 ± 20.8cP < .01 vs WT treated with DSS at the same time point.a Data are mean ± SEM of the number of BrdU-positive epithelial cells per 2 mm2 of colonic mucosa. N = 3 mice per group.b P < .01 vs WT control.c P < .01 vs WT treated with DSS at the same time point. Open table in a new tab The results obtained using DSS were confirmed with the model of TNBS-induced colitis. As detailed in Table 2, WT mice treated with TNBS developed severe inflammation, whereas KO mice were protected from TNBS-induced colitis. In addition, production of the proinflammatory cytokines IL-6 and interferon γ (IFN-γ) by colon cultures was significantly reduced in KO compared with WT mice treated with TNBS (Table 2). Thus, absence of APN led to decreased colonic inflammation associated with reduced cytokine production and preservation of epithelial cell proliferation in 2 models of chemically induced colitis.Table 2Clinical Score and Colonic Cytokine Production in WT and KO Mice Treated With TNBSTreatmentClinical scoreaData are mean ± SEM of clinical scores at day 16.IL-6bData are mean ± SEM of IL-6 and IFN-γ levels (pg/mL) in colon culture supernatants.IFN-γWT vehicle2.8 ± 0253 ± 8431 ± 17KO vehicle0 ± 058 ± 1961 ± 7WT TNBS7.5 ± 0.4949 ± 2371414 ± 589KO TNBS1.0 ± 0.4cP < .01 vs WT TNBS. N = 7 mice per group.117 ± 19cP < .01 vs WT TNBS. N = 7 mice per group.237 ± 93cP < .01 vs WT TNBS. N = 7 mice per group.a Data are mean ± SEM of clinical scores at day 16.b Data are mean ± SEM of IL-6 and IFN-γ levels (pg/mL) in colon culture supernatants.c P < .01 vs WT TNBS. N = 7 mice per group. Open table in a new tab To evaluate whether APN is present in colonic mucosa, we measured APN in colon homogenates as well as colon culture supernatants. As shown in Figure 2A, APN messenger RNA (mRNA) was expressed in the colon of both control and DSS-treated WT mice. We used quantitative PCR to compare the expression of APN mRNA in colon and adipose tissue. As shown in Figure 2B, mRNA APN levels in colonic tissue obtained from either control or DSS-treated WT mice were approximately 60% of expression observed in adipose tissue. Although, as expected, mRNA for APN was undetectable in the colon of KO mice, thus underlying the specificity of the primers used, comparable amounts of APN protein were detectable in colon homogenates of WT mice and of KO mice injected with exogenous APN (50 pg/mL in WT and 30 pg/mL in APN-injected KO mice). As expected, no immunoreactivity for APN was observed in homogenates obtained from KO mice that had not received exogenous APN, indicating that the assay used was specific for APN. When colon culture supernatants were evaluated for APN content, a significant release of APN from colonic tissue was observed in cultures of both DSS-treated WT and DSS-treated KO-APN mice, whereas colon from control (non-DSS) mice did not release APN, although it was positive for tissue-associated APN (Figure 2C). The combination of mRNA and protein data suggests that, although APN can be produced in the colon, circulating or luminal APN associates with colonic mucosa and can be released in the presence of inflammation. Measurement of APN in the intestinal lumen indicated that APN was detectable in the lumen of the colon but not in the small intestine in both WT and KO-APN mice (colonic APN was approximately 1 ng/mL in WT and 0.5 ng/mL in KO-APN). As a negative control, APN was not detectable in any samples obtained from KO mice that had not received exogenous APN. Fractionation of APN obtained from serum and colon culture supernatants was compared to evaluate the relative presence of high vs middle/low molecular weight complexes. As indicated in Table 3, the percentage distribution of APN was comparable in each of the samples analyzed. To localize APN in colonic mucosa, immunohistochemistry was performed. In non-DSS-treated WT mice and KO-APN mice, APN was detected selectively on the luminal surface of the colon and appeared to be associated with epithelial cells (Figure 2D). In contrast, in DSS-treated WT and DSS-treated KO-APN mice, immunoreactivity for APN was expressed widely in mucosal and submucosal layers. As expect
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