Tissue Eosinophilia in a Mouse Model of Colitis Is Highly Dependent on TLR2 and Independent of Mast Cells
2011; Elsevier BV; Volume: 178; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2010.11.041
ISSN1525-2191
AutoresEric J. Albert, Jon Duplisea, Wojciech Dawicki, Ian D. Haidl, Jean S. Marshall,
Tópico(s)Asthma and respiratory diseases
ResumoThe mechanisms initiating eosinophil influx into sites of inflammation have been well studied in allergic disease but are poorly understood in other settings. This study examined the roles of TLR2 and mast cells in eosinophil accumulation during a nonallergic model of eosinophilia-associated colitis. TLR2-deficient mice (TLR2−/−) developed a more severe colitis than wild-type mice in the dextran sodium sulfate (DSS) model. However, they had significantly fewer eosinophils in the submucosa of the cecum (P < 0.01) and mid-colon (P < 0.01) than did wild-type mice after DSS treatment. Decreased eosinophilia in TLR2−/− mice was associated with lower levels of cecal CCL11 (P < 0.01). Peritoneal eosinophils did not express TLR2 protein, but TLR2 ligand injection into the peritoneal cavity induced local eosinophil recruitment, indicating that TLR2 activation of other cell types can mediate eosinophil recruitment. After DSS treatment, mast cell-deficient (KitW-sh/W-sh) mice had similar levels of intestinal tissue eosinophilia were observed as those in wild-type mice. However, mast cell-deficient mice were partially protected from DSS-induced weight loss, an effect that was reversed by mast cell reconstitution. Overall, this study indicates a critical role for indirect TLR2-dependent pathways, but not mast cells, in the generation of eosinophilia in the large intestine during experimental colitis and has important implications for the regulation of eosinophils at mucosal inflammatory sites. The mechanisms initiating eosinophil influx into sites of inflammation have been well studied in allergic disease but are poorly understood in other settings. This study examined the roles of TLR2 and mast cells in eosinophil accumulation during a nonallergic model of eosinophilia-associated colitis. TLR2-deficient mice (TLR2−/−) developed a more severe colitis than wild-type mice in the dextran sodium sulfate (DSS) model. However, they had significantly fewer eosinophils in the submucosa of the cecum (P < 0.01) and mid-colon (P < 0.01) than did wild-type mice after DSS treatment. Decreased eosinophilia in TLR2−/− mice was associated with lower levels of cecal CCL11 (P < 0.01). Peritoneal eosinophils did not express TLR2 protein, but TLR2 ligand injection into the peritoneal cavity induced local eosinophil recruitment, indicating that TLR2 activation of other cell types can mediate eosinophil recruitment. After DSS treatment, mast cell-deficient (KitW-sh/W-sh) mice had similar levels of intestinal tissue eosinophilia were observed as those in wild-type mice. However, mast cell-deficient mice were partially protected from DSS-induced weight loss, an effect that was reversed by mast cell reconstitution. Overall, this study indicates a critical role for indirect TLR2-dependent pathways, but not mast cells, in the generation of eosinophilia in the large intestine during experimental colitis and has important implications for the regulation of eosinophils at mucosal inflammatory sites. Crohn's disease and ulcerative colitis are characterized by chronic relapsing inflammation of the gastrointestinal tract due to inappropriate responses of the mucosal immune system. A cellular infiltrate composed of several cell types, including eosinophils neutrophils, monocytes, and mast cells, is associated with intestinal inflammation in these inflammatory bowel diseases (IBD).1Xavier R.J. Podolsky D.K. Unravelling the pathogenesis of inflammatory bowel disease.Nature. 2007; 448: 427-434Crossref PubMed Scopus (3255) Google Scholar, 2Rothenberg M.E. Mishra A. Brandt E.B. Hogan S.P. Gastrointestinal eosinophils.Immunol Rev. 2001; 179: 139-155Crossref PubMed Scopus (238) Google Scholar, 3King T. Biddle W. Bhatia P. Moore J. Miner Jr, P.B. Colonic mucosal mast cell distribution at line of demarcation of active ulcerative colitis.Dig Dis Sci. 1992; 37: 490-495Crossref PubMed Scopus (79) Google Scholar, 4Nishida Y. Murase K. Isomoto H. Furusu H. Mizuta Y. Riddell R.H. Kohno S. Different distribution of mast cells and macrophages in colonic mucosa of patients with collagenous colitis and inflammatory bowel disease.Hepatogastroenterology. 2002; 49: 678-682PubMed Google Scholar Eosinophils are multifunctional leukocytes frequently mobilized during T helper 2 (Th2)-driven processes, such as allergic responses and parasitic helminth infections. In these contexts, eosinophils have been implicated in the pathogenesis of disease.5Weller P.F. Eosinophils: structure and functions.Curr Opin Immunol. 1994; 6: 85-90Crossref PubMed Scopus (118) Google Scholar, 6Gleich G.J. Loegering D.A. Immunobiology of eosinophils.Annu Rev Immunol. 1984; 2: 429-459Crossref PubMed Scopus (147) Google Scholar, 7Rothenberg M.E. Hogan S.P. The eosinophil.Annu Rev Immunol. 2006; 24: 147-174Crossref PubMed Scopus (1224) Google Scholar Eosinophil differentiation in the bone marrow and mobilization into the blood stream is regulated by a number of cytokines, including interleukin-5 (IL-5), granulocyte macrophage colony stimulating factor, and interleukin-3 (IL-3).8Lopez A.F. Sanderson C.J. Gamble J.R. Campbell H.D. Young I.G. Vadas M.A. Recombinant human interleukin 5 is a selective activator of human eosinophil function.J Exp Med. 1988; 167: 219-224Crossref PubMed Scopus (808) Google Scholar, 9Collins P.D. Marleau S. Griffiths-Johnson D.A. Jose P.J. Williams T.J. Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo.J Exp Med. 1995; 182: 1169-1174Crossref PubMed Scopus (573) Google Scholar, 10Dent L.A. Strath M. Mellor A.L. Sanderson C.J. Eosinophilia in transgenic mice expressing interleukin 5.J Exp Med. 1990; 172: 1425-1431Crossref PubMed Scopus (509) Google Scholar, 11Rothenberg M.E. Pomerantz J.L. Owen Jr, W.F. Avraham S. Soberman R.J. Austen K.F. Stevens R.L. Characterization of a human eosinophil proteoglycan, and augmentation of its biosynthesis and size by interleukin 3, interleukin 5, and granulocyte/macrophage colony stimulating factor.J Biol Chem. 1988; 263: 13901-13908Abstract Full Text PDF PubMed Google Scholar Further recruitment into tissues during an inflammatory response is orchestrated by a number of chemoattractants, including chemokines CCL5, CCL11, CCL7, and CCL3 and leukotrienes (LTs).7Rothenberg M.E. Hogan S.P. The eosinophil.Annu Rev Immunol. 2006; 24: 147-174Crossref PubMed Scopus (1224) Google Scholar The regulation of eosinophil recruitment has been best studied in allergic diseases such as atopic asthma. The role of eosinophils in intestinal inflammation is less well studied, except in the context of parasitic nematode infestation; however, there are well-documented intestinal inflammatory conditions in which tissue eosinophilia is a major feature, such as eosinophilic gastroenteritis and eosinophilic esophagitis.12Koga M. Fujiwara M. Hotta N. Matsubara T. Suzuki E. Furukawa S. MBP deposition in eosinophilic gastroenteritis.Allergy. 2000; 55: 985-986Crossref PubMed Scopus (5) Google Scholar, 13Bischoff S.C. Mayer J. Nguyen Q.T. Stolte M. Manns M.P. Immunnohistological assessment of intestinal eosinophil activation in patients with eosinophilic gastroenteritis and inflammatory bowel disease.Am J Gastroenterol. 1999; 94: 3521-3529Crossref PubMed Google Scholar An increase in eosinophil numbers has also been reported in subsets of IBD patients.14Carvalho A.T. Elia C.C. de Souza H.S. Elias P.R. Pontes E.L. Lukashok H.P. de Freitas F.C. Lapa e Silva J.R. Immunohistochemical study of intestinal eosinophils in inflammatory bowel disease.J Clin Gastroenterol. 2003; 36: 120-125Crossref PubMed Scopus (84) Google Scholar, 15Bischoff S.C. Wedemeyer J. Herrmann A. Meier P.N. Trautwein C. Cetin Y. Maschek H. Stolte M. Gebel M. Manns M.P. Quantitative assessment of intestinal eosinophils and mast cells in inflammatory bowel disease.Histopathology. 1996; 28: 1-13Crossref PubMed Scopus (233) Google Scholar Eosinophil numbers are known to be significantly increased in the mucosa and submucosa of the colon in C57BL/6 mice after dextran sodium sulfate (DSS) treatment, a well characterized and widely used model of inducible colitis.16Forbes E. Murase T. Yang M. Matthaei K.I. Lee J.J. Lee N.A. Foster P.S. Hogan S.P. Immunopathogenesis of experimental ulcerative colitis is mediated by eosinophil peroxidase.J Immunol. 2004; 172: 5664-5675PubMed Google Scholar, 17Stevceva L. Pavli P. Husband A. Matthaei K.I. Young I.G. Doe W.F. Eosinophilia is attenuated in experimental colitis induced in IL-5 deficient mice.Genes Immun. 2000; 1: 213-218Crossref PubMed Scopus (24) Google Scholar Moreover, this eosinophil recruitment was highly dependent on the activity of CCL11.16Forbes E. Murase T. Yang M. Matthaei K.I. Lee J.J. Lee N.A. Foster P.S. Hogan S.P. Immunopathogenesis of experimental ulcerative colitis is mediated by eosinophil peroxidase.J Immunol. 2004; 172: 5664-5675PubMed Google Scholar, 18Ahrens R. Waddell A. Seidu L. Blanchard C. Carey R. Forbes E. Lampinen M. Wilson T. Cohen E. Stringer K. Ballard E. Munitz A. Xu H. Lee N. Lee J.J. Rothenberg M.E. Denson L. Hogan S.P. Intestinal macrophage/epithelial cell-derived CCL11/eotaxin-1 mediates eosinophil recruitment and function in pediatric ulcerative colitis.J Immunol. 2008; 181: 7390-7399PubMed Google Scholar Toll-like receptors (TLR) are a family of innate immune pattern recognition receptors that recognize pathogen-associated molecular patterns. Several TLRs are expressed in the large intestine under normal physiological conditions and during inflammation.19Szebeni B. Veres G. Dezsofi A. Rusai K. Vannay A. Mraz M. Majorova E. Arató A. Increased expression of Toll-like receptor (TLR) 2 and TLR4 in the colonic mucosa of children with inflammatory bowel disease.Clin Exp Immunol. 2008; 151: 34-41Crossref PubMed Scopus (146) Google Scholar, 20Hart A.L. AL-Hassi H.O. Rigby R.J. Bell S.J. Emmanuel A.V. Knight S.C. Kamm M.A. Stagg A.J. Characteristics of intestinal dendritic cells in inflammatory bowel diseases.Gastroenterology. 2005; 129: 50-65Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 21Cario E. Rosenberg I.M. Brandwein S.L. Beck P.L. Reinecker H.C. Podolsky D.K. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors.J Immunol. 2000; 164: 966-972PubMed Google Scholar, 22Abreu M.T. Fukata M. Arditi M. TLR signaling in the gut in health and disease.J Immunol. 2005; 174: 4453-4460PubMed Google Scholar Activation of TLRs can lead to the production of proinflammatory cytokines and chemokines involved in the recruitment of leukocytes.23Fukata M. Michelsen K.S. Eri R. Thomas L.S. Hu B. Lukasek K. Nast C.C. Lechago J. Xu R. Naiki Y. Soliman A. Arditi M. Abreu M.T. Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G1055-G1065Crossref PubMed Scopus (423) Google Scholar, 24Rhee S.H. Keates A.C. Moyer M.P. Pothoulakis C. MEK is a key modulator for TLR5-induced interleukin-8 and MIP3alpha gene expression in non-transformed human colonic epithelial cells.J Biol Chem. 2004; 279: 25179-25188Crossref PubMed Scopus (76) Google Scholar TLR2-deficient animals have been shown to develop more severe colitis after DSS administration than their wild-type counterparts25Rakoff-Nahoum S. Paglino J. Eslami-Varzaneh F. Edberg S. Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.Cell. 2004; 118: 229-241Abstract Full Text Full Text PDF PubMed Scopus (3299) Google Scholar, 26Albert E.J. Marshall J.S. Aging in the absence of TLR2 is associated with reduced IFN-gamma responses in the large intestine and increased severity of induced colitis.J Leukoc Biol. 2008; 83: 833-842Crossref PubMed Scopus (24) Google Scholar as a result of a dysregulated interaction with intestinal microflora and altered epithelial permeability characteristics.27Cario E. Gerken G. Podolsky D.K. Toll-like receptor 2 controls mucosal inflammation by regulating epithelial barrier function.Gastroenterology. 2007; 132: 1359-1374Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar Stimulation of TLR2 by Mycobacterium bovis has also been shown to induce an eosinophilic infiltration into the pleural cavity that is highly dependent on IL-5, CCL11, and CCR3 (a CCL11 receptor).28D'Avila H. Almeida P.E. Roque N.R. Castro-Faria-Neto H.C. Bozza P.T. Toll-like receptor-2-mediated C-C chemokine receptor 3 and eotaxin-driven eosinophil influx induced by Mycobacterium bovis BCG pleurisy.Infect Immun. 2007; 75: 1507-1511Crossref PubMed Scopus (26) Google Scholar The role of TLR2 in eosinophil recruitment during DSS colitis and the potential role of eosinophils in the severe colitis seen in TLR2−/− mice have not been previously examined. Mast cells are strategically located at body sites that interface with the external environment, such as mucosal surfaces, where they have been shown to play an important role in host defense against parasitic and bacterial infection.29Barrett K.E. Metcalfe D.D. The mucosal mast cell and its role in gastrointestinal allergic diseases.Clin Rev Allergy. 1984; 2: 39-53PubMed Google Scholar, 30Malaviya R. Ikeda T. Ross E. Abraham S.N. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha.Nature. 1996; 381: 77-80Crossref PubMed Scopus (983) Google Scholar, 31Woodbury R.G. Miller H.R. Huntley J.F. Newlands G.F. Palliser A.C. Wakelin D. Mucosal mast cells are functionally active during spontaneous expulsion of intestinal nematode infections in rat.Nature. 1984; 312: 450-452Crossref PubMed Scopus (202) Google Scholar, 32Wei O.L. Hilliard A. Kalman D. Sherman M. Mast cells limit systemic bacterial dissemination but not colitis in response to Citrobacter rodentium.Infect Immun. 2005; 73: 1978-1985Crossref PubMed Scopus (54) Google Scholar, 33Supajatura V. Ushio H. Nakao A. Okumura K. Ra C. Ogawa H. Protective roles of mast cells against enterobacterial infection are mediated by Toll-like receptor 4.J Immunol. 2001; 167: 2250-2256PubMed Google Scholar, 34McDermott J.R. Bartram R.E. Knight P.A. Miller H.R. Garrod D.R. Grencis R.K. Mast cells disrupt epithelial barrier function during enteric nematode infection.Proc Natl Acad Sci USA. 2003; 100: 7761-7766Crossref PubMed Scopus (273) Google Scholar Mast cells have been suggested to play a proinflammatory role in models of colitis, and there is some evidence of mast cell activation in human IBD.15Bischoff S.C. Wedemeyer J. Herrmann A. Meier P.N. Trautwein C. Cetin Y. Maschek H. Stolte M. Gebel M. Manns M.P. Quantitative assessment of intestinal eosinophils and mast cells in inflammatory bowel disease.Histopathology. 1996; 28: 1-13Crossref PubMed Scopus (233) Google Scholar, 35Fox C.C. Lazenby A.J. Moore W.C. Yardley J.H. Bayless T.M. Lichtenstein L.M. Enhancement of human intestinal mast cell mediator release in active ulcerative colitis.Gastroenterology. 1990; 99: 119-124PubMed Google Scholar, 36Nolte H. Spjeldnaes N. Kruse A. Windelborg B. Histamine release from gut mast cells from patients with inflammatory bowel diseases.Gut. 1990; 31: 791-794Crossref PubMed Scopus (98) Google Scholar Mast cells are considered an important source of cytokines, chemokines, and other signaling factors that initiate the recruitment of eosinophils into sites of allergic inflammation.37Bischoff S.C. Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data.Nat Rev Immunol. 2007; 7: 93-104Crossref PubMed Scopus (488) Google Scholar, 38Bischoff S. Crowe S.E. Gastrointestinal food allergy: new insights into pathophysiology and clinical perspectives.Gastroenterology. 2005; 128: 1089-1113Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 39Theoharides T.C. Kempuraj D. Tagen M. Conti P. Kalogeromitros D. Differential release of mast cell mediators and the pathogenesis of inflammation [Erratum appeared in Immunol Rev 2007;219:204].Immunol Rev. 2007; 217: 65-78Crossref PubMed Scopus (335) Google Scholar Specifically, in the gastrointestinal tract mucosal mast cells can produce significant amounts of IL-5, CCL11, and LTC4 after IgE-mediated stimulation, making them key cells to study in disease processes associated with eosinophilia.40Lorentz A. Schwengberg S. Mierke C. Manns M.P. Bischoff S.C. Human intestinal mast cells produce IL-5 in vitro upon IgE receptor cross-linking and in vivo in the course of intestinal inflammatory disease.Eur J Immunol. 1999; 29: 1496-1503Crossref PubMed Scopus (71) Google Scholar Mast cells also express TLR2 and can produce cytokines and lipid mediators (eg, TNF, IL-5, IL-13, LTB4, and LTC4) in response to TLR2 activation.41Supajatura V. Ushio H. Nakao A. Akira S. Okumura K. Ra C. Ogawa H. Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity.J Clin Invest. 2002; 109: 1351-1359Crossref PubMed Scopus (445) Google Scholar, 42McCurdy J.D. Olynych T.J. Maher L.H. Marshall J.S. Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells.J Immunol. 2003; 170: 1625-1629PubMed Google Scholar, 43Olynych T.J. Jakeman D.L. Marshall J.S. Fungal zymosan induces leukotriene production by human mast cells through a dectin-1-dependent mechanism.J Allergy Clin Immunol. 2006; 118: 837-843Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar The role of mast cells has not been considered in detail in the DSS model of colitis. Other reports using the KitW/KitW-v (W/Wv) mast cell-deficient mouse model suggested that their absence was not associated with changes in disease severity. Mast cell-deficient mutant KitW-sh/W-sh mice containing the W-sash (Wsh) inversion mutation do not have many of the additional defects present in W/Wv mice.44Grimbaldeston M.A. Chen C.C. Piliponsky A.M. Tsai M. Tam S.Y. Galli S.J. Mast cell-deficient W-sash c-kit mutant KitW-sh/W-sh mice as a model for investigating mast cell biology in vivo.Am J Pathol. 2005; 167: 835-848Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar For example, there are normal levels of TCRγδ intraepithelial lymphocytes, making Wsh mice a much better model for studying the role of mast cells in eosinophil recruitment in the large intestine. The present study examined the role of TLR2 and mast cells in driving tissue eosinophilia during intestinal inflammation induced by DSS in mice and revealed an unexpected pivotal role for TLR2 in this process. All mice were 8–12 weeks of age. TLR2−/− mice backcrossed more than 10 generations onto a C57BL/6 background were kindly provided by Dr. S. Akira (Osaka University, Osaka, Japan). Mast cell-deficient B6.Cg-KitW-sh/HNihrJaeBsmJ mice and matched C57BL/6 control mice were obtained from Jackson Laboratory (Bar Harbor, ME). Animals were housed under specific pathogen-free conditions and were allowed free access to regular water and food. Because our previous studies45Albert E.J. Sommerfeld K. Gophna S. Marshall J.S. Gophna U. The gut microbiota of toll-like receptor 2-deficient mice exhibits lineage-specific modifications.Environ Microbiol Rep. 2009; 1: 65-70Crossref PubMed Scopus (11) Google Scholar illustrated that cohousing of littermates will not normalize the intestinal microbiota of TLR2-deficient mice, for this study we used mice that were not co-housed from birth (except that all mice from Jackson Laboratory were housed together with other strains in the Animal Care facility at Dalhousie University for a minimum of 2 weeks before use). All experiments were conducted in accordance with the guidelines of the Canadian Council on Animal Care (local protocol no. 03-102). Bone marrow-derived mast cells (BMMCs) were generated from C57BL/6 mice according to the method of Tertian et al.46Tertian G. Yung Y.P. Guy-Grand D. Moore M.A. Long-term in vitro culture of murine mast cells.I. Description of a growth factor-dependent culture technique. J Immunol. 1981; 127: 788-794Google Scholar The Wsh mice were reconstituted as previously described.47Nakano T. Sonoda T. Hayashi C. Yamatodani A. Kanayama Y. Yamamura T. Asai H. Yonezawa T. Kitamura Y. Galli S.J. Fate of bone marrow-derived cultured mast cells after intracutaneous, intraperitoneal, and intravenous transfer into genetically mast cell-deficient W/Wv mice.Evidence that cultured mast cells can give rise to both connective tissue type and mucosal mast cells. J Exp Med. 1985; 162: 1025-1043Google Scholar Briefly, 7.5 × 106 mature BMMC (> 98% pure mast cells) were injected intravenously into the tail vein of 8- to 12-week-old Wsh mice. Mice were allowed to mature in vivo for a minimum of 16 weeks to allow reconstitution of the gastrointestinal tract. Effective mast cell reconstitution was confirmed by histological analysis in Carnoy's-fixed, Toluidine Blue-stained paraffin sections. Mice were between 28 and 30 weeks of age when used in DSS colitis experiments. Mice received 3% (w/v) DSS (mol. wt. = 36–50 kDa; ICN Biomedicals, Aurora, OH) dissolved in sterile, distilled water ad libitum on days 0–5, followed by 5 days of regular drinking water. A similar DSS treatment protocol had been used by several groups previously.23Fukata M. Michelsen K.S. Eri R. Thomas L.S. Hu B. Lukasek K. Nast C.C. Lechago J. Xu R. Naiki Y. Soliman A. Arditi M. Abreu M.T. Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G1055-G1065Crossref PubMed Scopus (423) Google Scholar, 25Rakoff-Nahoum S. Paglino J. Eslami-Varzaneh F. Edberg S. Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.Cell. 2004; 118: 229-241Abstract Full Text Full Text PDF PubMed Scopus (3299) Google Scholar, 27Cario E. Gerken G. Podolsky D.K. Toll-like receptor 2 controls mucosal inflammation by regulating epithelial barrier function.Gastroenterology. 2007; 132: 1359-1374Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar Mice were weighed daily. Cecal, mid-colon, and lung samples were taken for histology and tissue sonication on days 0, 5, 7, and 10 of treatment. Peripheral blood samples were also taken from selected animals before and after treatment. This protocol yields maximal weight loss and histological damage at days 7–8 for control mice and at days 10–11 in TLR2-deficient animals.26Albert E.J. Marshall J.S. Aging in the absence of TLR2 is associated with reduced IFN-gamma responses in the large intestine and increased severity of induced colitis.J Leukoc Biol. 2008; 83: 833-842Crossref PubMed Scopus (24) Google Scholar The large intestine was removed free of fatty tissue and mesenteric lymph nodes. The entire colon length was measured from the cecum to the rectum. Cecal and mid-colonic portions of the large intestine were opened longitudinally, rinsed briefly in cold PBS, and further divided into two segments. One segment was frozen and saved for sonication, and the other half was fixed in 10% neutral buffered formalin and paraffin-embedded. All samples for cytokine content analysis were placed in PBS at a constant ratio to their weights, sonicated, and centrifuged (2300 g for 10 minutes), with supernatants collected for cytokine assays. Paraffin-embedded tissue sections were cut 5 μm thick and stained with hematoxylin and eosin for general histology or with Congo Red to identify eosinophils before evaluation. Five-micron sections of paraffin-embedded, formalin-fixed tissue were stained for 1 hour in 1% alcoholic Congo Red solution (1.0 g Congo Red, 50 ml ethanol, 50 ml distilled water). Slides were then placed in Mayer's hematoxylin for 2 minutes, followed by a rinse with tap water. Slides were blued in Scott's tap water substitute and then rinsed again with tap water. Finally, the slides were dehydrated, cleared, and mounted. The resulting stain showed eosinophil granules as bright red and nuclei as blue. Entire cecal and mid-colonic sections tissue sections were coded and assessed in a blinded manner. Histological damage was scored according to a previously described protocol.26Albert E.J. Marshall J.S. Aging in the absence of TLR2 is associated with reduced IFN-gamma responses in the large intestine and increased severity of induced colitis.J Leukoc Biol. 2008; 83: 833-842Crossref PubMed Scopus (24) Google Scholar Briefly, sections were scored based on three criteria; inflammatory cell infiltration (0–3), tissue damage (0–5), and edema (0–2). For colon length measurements, the entire large intestine was removed and length was measured from the proximal end of the cecum to the anus. Eosinophilia after DSS treatment was most pronounced in the submucosal area of the large intestine. In both cecal and colonic samples, the number of eosinophils in the submucosa was counted per area (20,000 μm2) of submucosa in Congo Red-stained sections, based on red granule staining. Ten random fields were counted per section. C57BL/6 mice were left untreated or injected with 50 μL of either saline or saline containing 1 μg of a TLR2/TLR6 agonist, FSL-1 (EMC Microcollections, Tübingen, Germany). After 16 hours, the mice were euthanized and the peritoneal cells were harvested by i.p. injection and recovery of 5 ml PBS, 0.5% bovine serum albumin, 5 mmol/L EDTA. The cells were counted and analyzed by fluorescence-activated cell sorting for the expression of Siglec-F, F4/80, CD11b, and Gr-1 to identify eosinophils as Siglec-Fpos, F4/80lo, CD11blo/neg, and Gr-1neg (all antibodies from eBioscience, San Diego, CA). Cells were collected on a FACSCalibur or FACSAria system (BD Biosciences, San Jose, CA) and were analyzed with WinList version 5.0 software (Verity Software House, Topsham, ME). Five-micron sections of Carnoy's-fixed, paraffin-embedded tissue were stained for mast cells using an adaptation of the method of Strobel, Miller, and Ferguson.48Strobel S. Miller H.R. Ferguson A. Human intestinal mucosal mast cells: evaluation of fixation and staining techniques.J Clin Pathol. 1981; 34: 851-858Crossref PubMed Scopus (266) Google Scholar Toluidine Blue stain was dissolved in 1 N HCl to provide better discrimination from goblet cell background tissue staining within the large intestine. Venous blood was collected from the aorta of mice immediately after sacrifice. Total white blood cell counts were performed on a hemocytometer after dilution of blood 1:10 in crystal violet solution. Blood smears were prepared and stained with Diff Quick stain set (Dade Behring, Newark, NJ) according to the manufacturer's protocol. Smears were examined at ×400 magnification. Two hundred white blood cells were counted per smear, and differentiation of lymphocytes, monocytes, eosinophils, and neutrophils was performed based on nuclear morphology and cytoplasmic staining properties. Enzyme-linked immunosorbent assays (ELISAs) were performed on cell-free supernatants according to the manufacturers' protocols. Detection of IL-5 was performed using a DuoSet mouse IL-5 ELISA development system (R&D Systems, Minneapolis, MN) (sensitivity:31.25 pg/ml). Amplification of the IL-5 ELISA was performed using a commercial ELISA amplification system (Invitrogen, Carlsbad, CA). Eotaxin ELISA was performed on cell-free supernatants using a Quantikine Mouse eotaxin immunoassay kit (R&D Systems) according to the manufacturer's protocol (sensitivity was 15.6 pg/ml). In general, cytokine content results were evaluated on a per-gram wet-weight basis. Protein content assays were also performed on a substantial subset of colon and cecum tissue samples from both control and inflamed mouse intestine, and cytokine contents were also calculated using this parameter. These values were highly correlated (P < 0.001) with the wet weight values. Results are reported as Mean +/− standard error of the mean (S.E.M.). Differences between TLR2−/− and wild-type mice and between Wsh and wild-type mice at a given time point were assessed using a one-way analysis of variance with a Bonferroni multiple comparison post hoc test. Comparisons of saline-injected versus FSL-1-injected mice and, where appropriate, individual groups of mice before and after DSS treatment, were performed using an unpaired t-test. A P-value of <0.05 was considered significant. According to several reports, TLR2−/− mice develop more severe intestinal inflammation than wild-type mice when treated with 3% DSS (w/v) for 5 days, followed by an additional 5 days of regular water administration.25Rakoff-Nahoum S. Paglino J. Eslami-Varzaneh F. Edberg S. Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.Cell. 2004; 118: 229-241Abstract Full Text Full Text PDF PubMed Scopus (3299) Google Scholar, 26Albert E.J. Marshall J.S. Aging in the absence of TLR2 is associated with reduced IFN-gamma responses in the large intestine and increased severity of induced colitis.J Leukoc Biol. 2008; 83: 833-842Crossref PubMed Scopus (24) Google Scholar We examined eosinophil recruitment in the cecums and mid-colons of TLR2−/− and wild-type mice after DSS treatment. Under normal physiological conditions, there were comparable levels of eosinophils in both the cecum and mid-colon of TLR2−/− and wild-type mice (Figure 1, A and D), and there were no significant differences between the number of peripheral blood eosinophils before treatment (data not shown). After 5 days of DSS treatment and 5 days of recovery (day 10), there was a significant increase in the number of submucosal eosinophils in the cecum of TLR2−/− and wild-type mice (P < 0.001) assessed by Congo Red staining (Figure 1, G and H). The
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