吞食蠕虫卵治疗溃疡性结肠炎的机理-----英文

DOI: 10.1126/scitranslmed.3001500 , 60ra88 (2010);2 Sci Transl Med , et al.Mara J. Broadhurst Infection in an Ulcerative Colitis Patient Trichuris trichiura T Cells Are Associated with Therapeutic + CD4+IL-22 http://stm.sciencemag.org/content/2/60/60ra88.full.html can be found at: and other services, including high-resolution figures,A complete electronic version of this article http://stm.sciencemag.org/content/suppl/2010/11/29/2.60.60ra88.DC1.html can be found in the online version of this article at: Supplementary Material http://stm.sciencemag.org/content/2/60/60ra88.full.html#ref-list-1 , 14 of which can be accessed free:cites 44 articlesThis article http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: article permission to reproduce this of this article or about obtaining reprintsInformation about obtaining is a registered trademark of AAAS. Science Translational Medicinerights reserved. The title NW, Washington, DC 20005. Copyright 2010 by the American Association for the Advancement of Science; all last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue (print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except theScience Translational Medicine o n D ec em be r 1 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m COL I T I S IL-22+ CD4+ T Cells Are Associated with Therapeutic Trichuris trichiura y o o e o fe o b an er m c L- in ity to modulate the immune system to enable their own survival pressive effects, and/or by helminths that can Because helminth infec- colitis when immuno- , 10), it is important to ate the balance between ucosa if live helminths n is typically character- duction of eosinophilia es, termed alternatively as interleukin-4 (IL-4) ing and tissue remodel- grity and mucosal heal- ing. IL-22, an IL-10 cytokine family member, also promotes wound s antimicrobial peptide e protective in animal induction of epithelial 7, 18). Although IL-22 ced by TH17 cells, it is cells can be induced in- refore, regulation of the be a potent avenue for Here, we have analyzed the colonic mucosa from a UC patient R E S EARCH ART I C L E o n D ec em be r 1 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m Diseases, University of California, San Francisco, CA 94158, USA. *These authors contributed equally to this work. as in clinical trials (7, 8) have suggested that helminth infection can prevent and/or treat IBD. A randomized placebo-controlled trial with Trichuris suis ova as therapy for UC indicated a therapeutic im- provement in a disease activity index (7), but the mechanism of lial cells (12, 13) and furthermore up-regulate and mucus production (14–17). IL-22 can b models of colitis, partially attributable to the wound healing and mucus production (13, 1 was originally described as a cytokine produ now recognized that IL-22 expression by TH dependently of IL-17 expression (19, 20). The production of TH2 cytokines and IL-22 could influencing the course of colitis. 1Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, CA 94143, USA. 2Department of Medical Parasitology, New York University Langone Medical Center, New York, NY 10010, USA. 3Division of Gastroenterology, Department of Medicine, Center for Colitis and Crohn’s Disease, University of Cali- fornia, San Francisco, CA 94143, USA. 4Sandler Center for Basic Research in Parasitic within mammalian hosts (5, 6). Studies of colitis in mice (6) as well healing, proliferation, and antiapoptotic pathways in intestinal epithe- cosa. Although the etiology of UC is poorly understood, it is believed that an impaired intestinal epithelial barrier, together with defects in mucosal immune regulation, favors the development of pathogenic T helper 17 (TH17) cells (1), probably in response to commensal micro- biota (2). Treatment of severe UC is usually through administration of immunosuppressive drugs, whose long-term use is limited by ad- verse side effects, including increased risk of opportunistic infection. Furthermore, up to 30% of patients develop disease refractory to treatment within 3 years of diagnosis, necessitating colectomy. IBD is most prevalent in Northern Europe, the United Kingdom, and North America (3) and historically rare in regions of endemic helminth infection such as Asia, Africa, and Latin America (3, 4). This has raised the hypothesis that helminths may protect against the path- ologic inflammation underlying IBD as a bystander effect of their abil- helminth-derived molecules with immunosup identification of biological pathways activated be targeted through conventional approaches. tions can themselves cause inflammation and regulatory networks are already disrupted (9 understand the molecular pathways that regul inflammation and immunity in the intestinal m are used as therapeutic agents. The immune response to helminth infectio ized by a TH2 response (5), including the in and of macrophages activated by TH2 cytokin activated macrophages. TH2 cytokines such and especially IL-13 contribute to wound heal ing (11) and may also promote epithelial inte Ulcerative colitis (UC), a major form of inflammatory bowel disease (IBD), is characterized by chronic inflammation of the colonic mu- this phenomenon could lead to the development of new therapeutics, for example, through infection with live helminths, identification of in an Ulcerative Colitis Patient Mara J. Broadhurst,1* Jacqueline M. Leung,2* Vikram Kash Uma Mahadevan,3 James H. McKerrow,4 P’ng Loke2† (Published 1 December 2010; Volume 2 Issue 60 60ra88) Ulcerative colitis, a type of inflammatory bowel disease, is less c infections, suggesting that helminth colonization may have the p in inflammatory bowel diseases. Indeed, therapeutic effects of exp ported in both animal models and clinical trials. Here, we provide a c of dynamic changes in the intestinal mucosa of an individual who in his symptoms of ulcerative colitis. Tissue with active colitis had a pr cells that produced the inflammatory cytokine interleukin-17 (IL-17) healing. After helminth exposure, the disease went into remission, the mucosa. Genes involved in carbohydrate and lipid metabolism w whereas tissues with active colitis showed up-regulation of proinfla CHI3L1. Therefore, T. trichiura colonization of the intestine may redu cell hyperplasia and mucus production through TH2 cytokines and I logical effects of helminth infection may lead to new therapies for INTRODUCTION †To whom correspondence should be addressed. E-mail: png.loke@nyumc.org www.Scien Infection ap,2 Joseph M. McCune,1 mmon in countries endemic for helminth tential to regulate intestinal inflammation rimental helminth infection have been re- mprehensive cellular and molecular portrait cted himself with Trichuris trichiura to treat minent population of mucosal T helper (TH) ut not IL-22, a cytokine involved in mucosal d IL-22–producing TH cells accumulated in e up-regulated in helminth-colonized tissue, matory genes such as IL-17, IL-13RA2, and e symptomatic colitis by promoting goblet 22. Improved understanding of the physio- flammatory bowel diseases. action was not characterized. A more complete understanding of who obtained symptomatic relief after infecting himself with the nem- ceTranslationalMedicine.org 1 December 2010 Vol 2 Issue 60 60ra88 1 to f u ig g c e Fig. 1. Overview of clinical course and analyses. (A) Time course of UC disease severity (red showing s g in ys d bi n ra 2 e a R E S EARCH ART I C L E o n D ec em be r 1 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m colitis and yellow showing remission) in relation to T. trichiura infection, biop biopsy tissue by transcriptional profiling and flow cytometry. The subject in 2004 and again in August 2008. Histopathology slides of biopsies collected 2005 were reviewed. In January 2007, biopsies were collected for RNA anal When the subject reverted to pan-colitis in July 2008, biopsies were collecte analysis. In March 2009, a colonoscopy demonstrated mucosal healing and for flow cytometry and RNA analysis. (B) Examples of gross pathology see tion. In 2007, worms were observed in the cecum, ascending colon, and t sigmoid colon was normal and the rectum exhibited signs of proctitis. In only in the ascending colon and not in the transverse colon; however, th hibited signs of severe colitis. In 2009, worms were mainly observed in the mucosa was observed in the remainder of the colon with few signs of infla www.Scien the previous colonoscopy (Fig. 2C and fig. S2). The histopathology of the sig- moid colon was essentially unchanged from 2005. Consistent with the flare of proctitis, macroscopic signs of colitis were apparent in the rectum (Fig. 2G). One biopsy contained a predominantly neu- trophilic infiltrate in the lamina propria, with cryptitis, a crypt abscess, and several prominent lymphoid aggregates (Fig. y collection, and analyses of ested parasite ova twice in August 2003 and October is during a flare of proctitis. for flow cytometry and RNA opsies were again collected upon endoscopic examina- nsverse colon, whereas the 008, worms were observed remainder of the colon ex- scending colon, and intact mmation. ceTranslationalMedicine.org 1 pathology of UC in the ection rse of a 35-year-old man diagnosed with . S1). His initial disease was severe and ents, mercaptopurine, and high-dose ste- olon had extensive ulceration of the mu- pithelial-lined mucosal glands remaining (Fig. 2A and fig. S2). Neutrophils heavily infiltrated the glands (Fig. 2B) and the surrounding lamina propria, forming crypt abscesses. In early 2004, the immu- nosuppressant cyclosporine or colectomy was advised. Instead, the patient chose to infect himself with T. trichiura eggs ob- tained in Thailand. He ingested 500 in vitro–germinated eggs in late 2004 and an additional 1000 eggs 3 months later. His symptoms improved in the follow- ing months, and by mid-2005, he was symptom-free and required no treatment for UC. He periodically took 5-aminosalicylates either topically or by mouth to control symptomatic flares, although he was gen- erally not under any medical therapy. In October 2005, T. trichiura worms (Fig. 2C) were observed in the cecum, as- cending colon, and transverse colon. Despite a prominent infiltration of eo- sinophils in the lamina propria of these tissues, the mucosa was intact with no significant epithelial cell loss and the glands appeared normal (Fig. 2C and fig. S2). In contrast to 2003, no ulceration or neutrophil infiltration was observed in the sigmoid colon, and glands demon- strated moderate goblet cell hyperplasia and mucus hypersecretion (Fig. 2D). A brief flare of symptoms in 2007 war- ranted a colonoscopy. The ascending co- lon continued to harbor a heavy worm burden (Fig. 1B and fig. S1). This tissue showed no ulceration, with normal glands and many macrophages, eosinophils, and plasma cells (Fig. 2, E and F), similar to atode parasite Trichuris trichiura. It is estimated that almost a billion people worldwide are infected with T. trichiura, with the highest prev- alence in Central Africa, southern India, and Southeast Asia (4, 21). In contrast to T. suis ova, this human parasite establishes chronic, not transient, colonization. Genome-wide transcriptional profiling, flow cytometric evaluation of the immune response, and histological analyses were performed on biopsy samples collected during active colitis and during stable disease remission associated with helminth infection. RESULTS Clinical course and his setting of T. trichiura in We followed the disease co UC in 2003 (Fig. 1 and f refractory to mesalamine a roids. In 2003, his sigmoid cosal epithelium, with few 2G). In contrast, other biopsies showed December 2010 Vol 2 Issue 60 60ra88 2 bars, 100 mm; Red scale bars, 50 mm. were created for cytokine positive cells and used to divide the cytokine- producing cells into distinct populations corresponding to the patterns of R E S EARCH ART I C L E www.Scien o n D ec em be r 1 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m cytokines that they are producing. These results were then graphed using the SPICE software to generate the pie charts representing the proportion of cytokine-expressing CD4+ T cells that express combinations of IL-17, IL-22, IFN-g, and/or IL-4. Cells that are not producing any of these cytokines are not represented in the pie charts. (B) Flow cytometry bivariate contour plots showing the frequencies of CD4+ T cells expressing IL-17, IL-22, IFN-g, and IL-4 from biopsy samples taken from the sigmoid colon and ascending colon in 2008 and 2009. (C) Mucus production in the sigmoid colonwas visualized Fig. 2. Distinct inflammatory infiltrates characterize colitis-affected and helminth-exposedmucosal tissues. Tissue sectionsof colon biopsies stained with hematoxylin and eosin. (A) Sigmoid colon in 2003 showed signs of severe colitis including ulcerated epithelium (UE) and crypt abscesses (CA). (B) High- powered view of the sigmoid colon in 2003 showing prominent neutrophils (N), infiltration of glands, and a crypt abscess (CA). (C) Ascending colon in 2005 with cross section of a T. trichiura worm (W), prominent eosinophils (E), and plasma cells (P), as well as goblet cell (G) hyperplasia. (D) Sigmoid colon in 2005 showed restoration of glands (gl) and goblet cells (G). (E) Ascending colon in 2007 with cross section of a T. trichiuraworm, with prominent eosino- phils and goblet cells. (F) High-power view of the ascending colon in 2007 showing prominent eosinophilia and goblet cells. (G) Proctitis in the rectum in 2007 showed lymphoid aggregates (LA) and neutrophil infiltration into the glands. (H) Severe colitis in the sigmoid colon in 2008 showing loss of glands and ulcerated epithelia and crypt abscess. (I) High-power view of sigmoid colon in 2008 showingpronouncedneutrophil infiltration. (J) Sigmoid colon in 2009 showed restoration of glands and goblet cells. Black scale Fig. 3. Induction of IL-22 expression in TH cells of the sigmoid colon is associated with restoration of mucus production. Lymphocytes from colon biopsies collected in 2008 and 2009 were analyzed by flow cytometry for intracellular cytokine expression after a 5-hour PMA-ionomycin stimu- lation ex vivo. (A) Visualization of the combinations of cytokines expressed by CD4+ T cells from colon biopsies with pie charts in which each slice represents a different cytokine combination. The flow cytometric gating strategy used to generate these charts is shown in fig. S3. Boolean gates minimal infiltration of the lamina propria, intact epithelium, and goblet cell hyperplasia. After 3 years of nearly complete disease remission, the patient’s symptoms began to deteriorate in mid-2008, paralleling a decline in stool egg counts from an extremely high number (>15,000 eggs per gram) to more moderate numbers (<7000 eggs per gram). There was active colitis in both the ascending and the sigmoid colons (Fig. by the periodic acid–Schiff (PAS) stain. Scale bars, 50 mm. ceTranslationalMedicine.org 1 December 2010 Vol 2 Issue 60 60ra88 3 ments collected during colonoscopy. After culture, cells were assayed by flow cytometry for intracellular cytokine expression after a 5-hour PMA- R E S EARCH ART I C L E o n D ec em be r 1 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m ionomycin stimulation. (A and B) Percentage of CD4+ T cells that produce cytokines in response to T. trichiura antigen stimulation compared to cells cultured in media alone are shown as histograms (A) or bivariate flow cytometry contour plots (B). (C) Flow cytometry gating strategy to illus- trate an expansion of T. trichiura antigen-specific IL-4+ cells within both the IL-22+ and the IL-17+ CD4+ T cell subsets from PBMCs that were col- lected after reinfection (September 2008). Worm antigen was added at 100 mg/ml (high) or 25 mg/ml (low). SSC, side scatter. www.Scien epithelial layer (Fig. 2H). Ulceration and granulation tissue indicated that there was chronic inflammation in this tissue. The patient chose to infect himself again, this time with 2000 T. trichiura eggs. His symptoms improved in the following months, and he required no other medication. He had another colonoscopy in early 2009. Large numbers of worms were observed in the ascending colon (Fig. 1B), and beyond an eosinophilic infiltrate in the lamina propria, no signs of colitis were present. The sigmoid colon still demon- strated mild colitis, with a few scattered neutrophils in the lamina propria and minor crypt distortion. However, goblet cell numbers were restored and no ulceration was noted (Fig. 2J). Thus, renewed T. trichiura col- onization was again associated with improvement in histopathologic findings, including normalization of tissue architecture and recovery of epithelial integrity. Characterization of TH cell responses in the colonic mucosa Biopsies collected during the 2008 and 2009 colonoscopies were ana- lyzed by flow cytometry for intracellular IL-17, IL-22, IL-4, interferon-g (IFN-g), and tumor necrosis factor–a (TNFa) (fig. S3) production. We examined the relative proportions of monocytokine- and polycytokine- producing CD4+ TH cells (Fig. 3A). In 2008, during the relapse of symp- tomatic colitis, 70% of cytokine-producing CD4+ TH cells from the severely inflamed sigmoid colon expressed only IL-17 (Fig. 3 and fig. S3), whereas less than 1% expressed IL-22. In contrast, biopsies from the ascending and transverse colon, in which epithelial integrity and gland structure remained intact, had more IL-22+ cells (Fig. 3 and fig. S3). Analysis of mucosal TH cell responses in 2009, during the period of remission after reinfection with T. trichiura, revealed that IL-17 expression by TH cells in the sigmoid colon was comparable to that of 2008 (Fig. 3A). Furthermore, the IL-17 response was more prominent in the ascending and transverse colon in 2009 than in 2008. However, a significant population of IL-22+ TH cells was now observed in the sigmoid colon (Fig. 3, A and B), such that the proportion of IL-22+ TH cells was similar in the sigmoid and ascending colon. As expected, IL-4+ TH cells were also increased in the helminth-colonized ascending colon (Fig. 3 and fig. S3). Thus, despite the continued presence of CD4+ IL-17+ TH cells, symptomatic remission asso- ciated with a reduction in infiltrating neutrophils and restoration of tissue architecture was characterized by the presence of CD4+ IL-22+ TH cells. Because the protective effect of IL-22 in mouse models of colitis is linked with the stimulation of mucus production by goblet cells (17), we used the periodic acid–Schiff (PAS) stain to determine that although virtually no mucus was detectable in the sigmoid colon in 2008, am- ple mucus production was present in 2009 (Fig. 3C). A role for TNFa in UC pathogenesis has been suggested by the positive outcomes of several clinical trials with the anti-TNF antibody infliximab (22). TNFa is expressed by several types of immune cells, notably activated macrophages and T cells. Although we found a higher frequency of TNFa+ TH cells in colitis-affected tissue compared to helminth-exposed tissue in 2008 (fig. S3), remission was not associated with the suppres- sion of TNFa responses. The inflammatory response to reinfection with T. trichiura was characterized by a high frequency of TNFa+ TH cells (fig. S3). In summary, active colitis was associated with monocytokine-producing CD4+ IL-17+ TH cells, whereas helminth colonization and disease remission were characterized by the presence of IL-22+ TH cells in the colonic mucosa. High frequencies of mucosal IL-17+ and TNFa+ TH cells were associated with active colitis in 2008, Fig. 4. IL-4+