REVIEW URRENT C OPINION
Chronic mucosal inflammation/inflammatory bowel disease-like inflammation after intestinal transplantation: where are we now? Cal S. Matsumoto, Michael A. Zasloff, and Thomas M. Fishbein
Purpose of review The purpose of this review is to highlight the similarities between inflammatory bowel disease and the state of the intestine allograft after transplantation. Recent findings The mutant nucleotide-binding oligomerization protein 2 (NOD2) gene, which encodes for an intracellular protein that serves as an innate immune system microbial sensor in macrophages, dendritic cells, and certain intestinal epithelial cells, has been recognized as a risk factor in Crohn’s disease. Similarly, recent studies have also highlighted the contribution the NOD2 mutation may have on intestinal failure itself. More specifically, in intestinal transplant recipients with the NOD2 mutation, the discovery of the reduced ability to prevent bacterial clearance, increased enterocyte stress response, and failure of key downstream expression of important cytokines and growth factors have been implicated as major factors in intestinal transplant outcomes, namely graft loss and septic death. Treatment strategies with anti tumor necrosis factor (TNF) a, similar to inflammatory bowel disease, have been employed in intestinal transplantation with promising results. Summary In intestinal transplantation, there is evidence that the classical alloimmunity pathways that lead toward graft dysfunction and eventual graft loss may, in fact, be working in concert with a disordered innate immune system to produce a state of chronic inflammation not unlike that seen in inflammatory bowel disease. Keywords inflammatory bowel disease, intestine transplant, NOD2
INTRODUCTION The human intestinal tract exists in a constant state of balance between the destructive capabilities of microbes and the active protective mechanisms of the gut epithelium. Any condition that affects the fine balance of the gut protective mechanisms has the potential to disrupt the chemical and biological immune interfaces of the gut epithelium and produce a disease state. Inflammatory bowel disease (IBD), of which Crohn’s disease and ulcerative colitis are the two most common forms, has origins that are most likely multifactorial in nature [1]. Genetic and environmental factors seem to play an important role, and recent developments have further provided substantial evidence of a dysregulated immune response in a compromised gut epithelium as a major contributor to this disease state [2,3]. A similar state of a compromised gut epithelium is observed after transplantation of the human www.co-transplantation.com
intestine. From the very onset, the gut epithelium is rendered an injury due to the brain death condition of the donor, procurement, and preservation process. Obligate ischemia and reperfusion injury contribute toward epithelial injury, and from then, a lifetime of chronic immunological assault ensues as the host relentlessly attempts allograft destruction, despite chronic immunosuppression. In areas of gut epithelial injury due to all the above factors, a weakened antimicrobial barrier can activate the innate immune response producing a MedStar Georgetown Transplant Institute, Georgetown University Hospital, Washington, DC, USA Correspondence to Cal S. Matsumoto, MD, MedStar Georgetown Transplant Institute, 2 Main, Georgetown University Hospital, 3800 Reservoir Rd., NW, Washington DC, USA. Tel: +1 202 444 3634; fax: +1 202 444 7304; e-mail: [email protected] Curr Opin Organ Transplant 2014, 19:276–280 DOI:10.1097/MOT.0000000000000077 Volume 19 Number 3 June 2014
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Chronic mucosal inflammation after transplantation Matsumoto et al.
KEY POINTS A significant positive association is observed in patients with intestinal failure and the NOD2 genotype mutation, a known risk factor for the development of Crohn’s disease. Recipients of an intestinal transplant with an NOD2 mutation have a higher risk of graft loss and septic death possibly due to a dysregulated innate immune system initiating an injurious inflammatory cascade like what is observed in inflammatory bowel disease. NOD2 mutation intestine transplant recipients have a reduced ability to prevent bacterial adherence, express a greater amount of stress cytokines, and have an impaired Paneth cell antimicrobial response. Intestine transplant recipients with ileal ulcerations have been observed to respond in a similar manner as patients with inflammatory bowel disease to anti-TNF-a therapy, suggesting a similar pathophysiologic mechanism.
scenario similarly observed in nontransplant patients with IBD. In addition, a genetic predisposition with faulty microbial sensoring mechanisms may play a similar role in intestinal transplantation as in IBD.
THE NOD2 POLYMORPHISM AND INFLAMMATORY BOWEL DISEASE-LIKE INJURY AFTER INTESTINAL TRANSPLANT The first description of the potential commonality of a dysregulated innate immune response in the transplanted human intestine and the disease state of IBD was described by Fishbein et al. [4]. In 30 intestinal transplant recipients, an intestinal transplant–IBD phenotype was described in 10% of cases, with gross features of graft ulcerations and a significantly decreased number of Paneth cells, the cells chiefly responsible for the innate immune response, on histological examination. In addition, ulceration healing was noted in 75% of cases with a mean response time of 6 weeks with the administration of anti-tumor necrosis factor (anti-TNF) therapy. Further studies by the same authors led to the discovery, in 2008, of the significant association between NOD2 polymorphisms and poor outcomes after intestinal transplantation [5]. The NOD2 gene encodes for an intracellular protein that serves as a microbial sensor in macrophages, dendritic cells, and certain intestinal epithelial cells, such as Paneth cells. Through these intestinal epithelial cells and gut-associated immune cells, the NOD2 protein promotes the expression of antimicrobial peptides (HD5, HD6), cytokines (TNF-a), and chemokines
(CX3CL1). Mutations of certain several alleles have been identified as functionally abnormal, with an increased risk for Crohn’s disease between two- and 40-fold, depending on the genotype of the individual [6,7]. In Fishbein’s analysis of intestinal transplant recipients with the NOD2 gene mutation, the likelihood of allograft failure was 97-fold higher than recipients with wild-type NOD2 loci. Additionally, in NOD2 mutant recipients, prior to observing an allograft rejection episode, a significant decrease in epithelial antimicrobial peptides human defensin 5 (HD5) and human b-defensin 2 (HBD2) was observed, despite normal bowel function and histology at the time of sampling. The authors conclude that the NOD2 mutant recipient of an intestinal transplant represents a significant risk factor for both rejection and death with the early event of this process involving the impaired expression of epithelium-derived antimicrobial peptides. Further evidence of the association of an NOD2 polymorphism resulting in a dysregulated innate immune system in intestinal failure (IF) patients as well as follow-up of certain subgroups after intestine transplant was reported by Ningappa et al. [8]. In an analysis of 60 Caucasian children with a primary intestine transplant, Ningappa specifically identified the NOD2 mutation at the R702W allele as a significant risk allele for the need of a combined liverintestine transplant as compared with 538 healthy controls of the same racial background (13.5 versus 3.6%, P ¼ 0.0007). The R702W allele was further identified as a significant risk allele associated with early rejection episodes (P ¼ 0.028) independent of other covariates in a linear regression analysis. Patient survival after liver-intestine transplant in those with the R702W allele was also significantly decreased (P ¼ 0.015) with half of the deaths resulting from septic events. The strong association of these NOD2 mutant IF patients requiring a liver-inclusive intestine transplant is best explained by the defect in the epithelial innate immune system contributing to a chronic epithelial bacterial invasion and portal endotoxemia which eventually leads to liver dysfunction and liver failure. In the posttransplant setting, the poor outcome associated with a recipient NOD2 polymorphism may represent a continuing susceptibility to inflammatory damage (which is an intrinsic characteristic of the recipient) or may be due to the potentiating effects of the inflammation on the graft alloimmunity process.
THE NOD2 POLYMORPHISM AND ASSOCIATION WITH INTESTINAL FAILURE Two recent studies have also highlighted the contribution of the NOD2 mutation may have on IF
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Small bowel transplantation &
itself. Schaffler et al. [9 ] genotyped 46 non-Crohn’s disease patients with IF and observed a significant increase in the frequency of the NOD2 allele mutation as compared with healthy controls (14 versus 6%, P ¼ 0.006). Despite the higher incidence of IF with the NOD2 mutation, this did not translate into an increased need for intestinal transplantation in their analysis, most likely due to the overall low incidence of intestinal transplants performed at their center. In an extension of the original analysis by Fishbein, Guerra et al. [10 ] analyzed the NOD2 allele frequency of 192 consecutive patients with IF as compared with 103 healthy controls. Overall incidence of NOD2 mutation in the IF group was significantly greater than healthy controls (26 versus 4.8%, P < 0.0001) and even with the exclusion of Crohn’s disease patients, the positive influence of NOD2 mutation status remained significant (18.8 versus 4.8%, P ¼ 0.001). &
EPITHELIAL CELL DYSREGULATION IN INFLAMMATORY BOWEL DISEASE AND AFTER INTESTINAL TRANSPLANT An association between intestinal epithelial cell barrier dysfunction and IBD has been well established in several independent studies. A critical factor in the pathogenesis of IBD is the disturbed antimicrobial defense provided by the intestine innate immune system. A conspicuous lack of Paneth cell a-defensins in ileal Crohn’s disease , both with the wild-type NOD2 phenotype and, more pronounced, in the NOD2 mutant phenotype has been established as a key element linking a dysfunctional intestinal innate immune system to IBD [11–14]. In Fishbein’s original work, a reduced expression of HD5 was observed in intestinal transplant recipients with the NOD2 mutation. Lough et al. [15 ] has further defined this work by observing not only reduced antimicrobial expression in NOD2 mutation–engrafted intestinal transplant recipients, but also a reduced ability to prevent bacterial adherence, a significant increased expression of CX3CL1, a stress cytokine produced by the injured enterocyte, and a failure of the NOD2 mutant intestinal transplant recipient to express key genes, such as Wnt5a, which regulate Paneth cell activation. Twenty-four non-Crohn’s disease recipients of an intestine transplant were evaluated, 12 with the NOD2 mutation and 12 with the wildtype NOD2 genotype. Bacterial adherence was examined with a probe (EUB338) targeting bacterial 16S rRNA by fluorescent in-situ hybridization methods. Consistent observations of relatively bacteria-free epithelial adherence was noted in the NOD2 wild-type recipients as compared with the NOD2 mutant recipients by averaged fluorescent intensity &&
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(P 0.05), leading to the speculation that this results as a consequence of the failure of Paneth cell function to produce effective levels of antimicrobial peptides in NOD2 mutant recipients. Further evidence of the downstream dysregulation of the epithelium in the NOD2 mutant recipients was also addressed by the authors. The cytokine CX3CL1, a recognized injury-related protein, was observed by semiquantitative analysis as well as by relative mRNA reverse transcription PCR to be significantly more expressed in the NOD2 mutant recipients, indicating a greater epithelial stress response, most likely from the overabundance of adherent pathogenic microbes, as described above. The most striking observation made in this report was the discovery that a myeloid cell of recipient origin, and which carried an NOD2 mutation appeared to be functionally defective. Unlike the comparable cell from NOD2 wild-type recipients, this cell failed to extend processes into the surface epithelial layer, suggesting it was no longer capable of monitoring the apical microenvironment. The NOD2 mutant myeloid cell also failed to express many cytokines and growth factors secreted by the NOD2 wild-type cell. These included Wnt5a, a growth factor known to stimulate expression of antimicrobial peptides and proteins, including HD5, within the Paneth cell. In NOD2 mutant recipients, lower secretions of Wnt5a as well as Wnt5a mRNA expression were observed as compared with NOD2 wild type (P 0.05). The finding that Wnt5a is secreted by a ‘sentinel’ myeloid cell within the intestinal lamina propria suggests that these cells coordinate with Paneth cell antimicrobial activity to match the threat of microbial invasion at the epithelial boundary. In addition, it is conceivable that this same ‘sentinel’ cell also plays a role in renewal rate of the small intestinal epithelium, since Wnt5a also stimulates the rate of stem cell cycling within the intestinal crypt. Furthermore, through its secretion of numerous cytokines, the same cell might also help orchestrate the community of immune cells within the lamina propria to effectively manage microbial invasion into that compartment. This evidence, from comparable intestine transplant recipients with differing NOD2 genotype status, may provide a provocative explanation to account for the increased morbidity and mortality associated with intestinal transplantation in general.
USE OF ANTI-TNF AGENTS IN INTESTINAL TRANSPLANT Perhaps the most compelling clinical evidence of the similar association between IBD and the Volume 19 Number 3 June 2014
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Chronic mucosal inflammation after transplantation Matsumoto et al.
intestinal transplant recipient comes in the form of the clinical response of these patients from the administration of TNF-a inhibitors. TNF-a has been firmly established as playing a pivotal role in the inflammatory response that is orchestrated in patients suffering from IBD. Through its proinflammatory signaling pathways, TNF-a upregulates other inflammatory cytokines and, among other nefarious activities, it induces epithelial cell apoptosis as well as a degradation of epithelial expression of tight junctions, further causing exacerbation of inflammation through a weakened mucosal barrier [16]. In patients with IBD, concentrations of TNF-a are elevated in the blood, intestinal mucosa, and stool [17]. TNF-a inhibitors, not surprisingly, have significantly altered the treatment landscape of IBD in the last decade with superior remission rates and improved mucosal healing in IBD than what had been observed with conventional medical therapy [18]. As reported by Pascher et al. with the successful treatment of two intestinal transplant recipients with Orthoclone (OKT3)-resistant ulcerative inflammation and similarly observed by Fishbein in 2006 with the healing of intestinal transplant ulceration in 75% of the cases, the use of anti-TNF-a agents and the similar outcomes as with IBD patients may imply that similar forces are behind the molecular pathophysiology of transplant graft ulceration and dysfunction [4,19]. The specific role in intestinal transplantation, however, remains less clear. Gerlach et al. [20] reported the use of Infliximab (Remicaide; Janssen Biotech (formerly Centocor Inc.), Horsham, PA, USA; ESSEX PHARMA GmbH, Munich, Germany) in nine intestine transplant patients: two with early onset OKT3-resistant acute cellular rejection (ACR) two with ulcerative distal graft inflammation, and five with late onset steroid-resistant ACR or OKT3-resistant ACR with chronic ulcerative graft changes. In eight of nine patients, Infliximab administration resulted in resolution of elevated serum TNF-a levels together with resolution of graft changes of either a chronic ulcerative nature or acute cellular injury. One patient who did not respond had most likely a significant humoral component injury; of which Th1-driven inflammatory antagonist may not play a role. Most striking was the reproducible success of Infliximab with late onset graft ulceration, which represents a more similar clinical setting; of which the successful use of Infliximab in IBD has been observed. A more recent report from De Greef et al. [21] describes the use of Infliximab for the treatment of refractory ACR in two pediatric intestinal transplant recipients. No chronic graft ulcerations were noted in these cases, and infliximab was used as a salvage therapy for
thymoglobulin-resistant rejection, possibly implicating TNF-a as a major mediator for ACR, as similarly observed by Gerlach. With limited data from the intestinal transplant experience, however, we can only postulate that the role played by antiTNF-a therapy has similar mechanisms to that of in IBD patients.
CONCLUSION The current extent of our knowledge regarding the actual pathophysiology of intestine allograft rejection remains limited. From an epidemiological standpoint, there is strong evidence that patients with IF itself may share certain immutable traits as with those suffering from IBD. Evidence from the field of IBD points to a dysregulation of the innate immune system, whether inherent or acquired, as the chief mediator of IBD pathology. In intestinal transplantation, there is evidence that the classical alloimmunity pathways that lead toward graft dysfunction and eventual graft loss may, in fact, be working in concert with a disordered innate immune system to produce a state of chronic inflammation not unlike that seen in IBD. Given the intricate immunologic and environmental variables inherent and unique to the human intestine, there may possibly be a multitude of pathways that lead to graft destruction and loss. Whether it is a disordered innate immune system that is a product of a classic human leukocyte antigen host–graft immunologic interaction or simply an inherent quality of certain high-risk recipient genotypes is a question that needs an answer with further research. The truth for intestinal transplantation most likely lies in-between as a combination that has been described previously as the third form of IBD. Acknowledgements None. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Danese S, Fiocchi C. Etiopathogenesis of inflammatory bowel diseases. World J Gastroenterol 2006; 12:4807–4812. 2. Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002; 347:417– 429. 3. Gersemann M, Wehkamp J, Stange EF. Innate immune dysfunction in inflammatory bowel disease. J Intern Med 2012; 271:421–428.
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Small bowel transplantation 4. Fishbein T, Novitsky G, Matsumoto C, et al. Innate immunity is altered after intestinal transplantation causing a new inflammatory bowel disease. Transplantation 2006; 82 (Supp 3):372. 5. Fishbein T, Novitskiy G, Mishra L, et al. NOD2-expressing bone marrowderived cells appear to regulate epithelial innate immunity of the transplanted human small intestine. Gut 2008; 57:323–330. 6. Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 2001; 411: 599–603. 7. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 2001; 411:603– 606. 8. Ningappa M, Higgs BW, Weeks DE, et al. NOD2 gene polymorphism rs2066844 associates with need for combined liver-intestine transplantation in children with short-gut syndrome. Am J Gastroenterol 2011; 106:157–165. 9. Scha¨ffler H, Schneider N, Hsieh CJ, et al. NOD2 mutations are associated & with the development of intestinal failure in the absence of Crohn’s disease. Clin Nutr 2013; 32:1029–1035. An analysis of the association between NOD2 mutation and intestinal failure. 10. Guerra JF, Zasloff M, Lough D, et al. Nucleotide oligomerization domain 2 & polymorphisms in patients with intestinal failure. J Gastroenterol Hepatol 2013; 28:309–313. A follow-up analysis confirming the association between intestinal failure and NOD2 mutation. 11. Wehkamp J, Koslowski M, Wang G, Strange EF. Barrier dysfunction due to distinct defensin deficiencies in small intestine and colonic Crohn’s disease. Mucosal Immunol 2008; 1 (s1):S67–74. 12. Cairo E. Commensal-innate immune miscommunication in IBD pathogenesis. Dig Dis 2012; 30:334–340.
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13. Wehkamp J, Schmid M, Fellermann K, Stange EF. Defensin deficiency, intestinal microbes and clinical phenotypes of Crohn’s disease. J Leukoc Biol 2005; 77:460–465. 14. Wehkamp J, Harder J, Weichenthal M, et al. NOD2 (CARD15) mutations in Crohn’s disease are associated with diminished mucosal alpha-defensin expression. Gut 2004; 53:1658–1664. 15. Lough D, Abdo J, Guerra-Castro J, et al. Abnormal CX3CR1þ lamina propria && myeloid cells from intestinal transplant recipients with NOD2 mutations. Am J of Transpl 2012; 12:992–1003. In-depth analysis of disordered and dysregulated innate immune system in NOD2 mutant intestinal transplant recipients. 16. Hanauer SB, Feagan BG, Lichtenstein G, et al. Maintenance infliximab for Crohn’s disease: the ACCENT I randomised trial. Lancet 2002; 359:1541– 1549. 17. Van Hogezand RA, Verspaget HW. The future role of antitumour necrosis factor-alpha products in the treatment of Crohn’s disease. Drugs 1998; 56:229–305. 18. Magro F, Portela F. Management of inflammatory bowel disease with infliximab and other antitumor necrosis factor alpha therapies. BioDrugs 2010; 24:3– 14. 19. Pascher A, Radke C, Dignass A, et al. Successful infliximab treatment of steroid and OKT3 refractory acute cellular rejection in two patients after intestinal transplantation. Transplantation 2003; 76:615. 20. Gerlach UA, Koch M, Muller HP, et al. Tumor necrosis factor alpha inhibitors as immunomodulatory antirejection agents after intestinal transplantation. Am J Transpl 2011; 11:1041–1050. 21. De Greef E, Avitzur Y, Grant D, et al. Infliximab as salvage therapy in paediatric intestinal transplant with steroid- and thymoglobulin-resistant late acute rejection. J Pediatr Gastroenterol Nutr 2012; 54:565–567.
Volume 19 Number 3 June 2014
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Chronic mucosal inflammation/inflammatory bowel disease-like inflammation after intestinal transplantation: where are we now? Cal S. Matsumoto, Michael A. Zasloff, and Thomas M. Fishbein
Purpose of review The purpose of this review is to highlight the similarities between inflammatory bowel disease and the state of the intestine allograft after transplantation. Recent findings The mutant nucleotide-binding oligomerization protein 2 (NOD2) gene, which encodes for an intracellular protein that serves as an innate immune system microbial sensor in macrophages, dendritic cells, and certain intestinal epithelial cells, has been recognized as a risk factor in Crohn’s disease. Similarly, recent studies have also highlighted the contribution the NOD2 mutation may have on intestinal failure itself. More specifically, in intestinal transplant recipients with the NOD2 mutation, the discovery of the reduced ability to prevent bacterial clearance, increased enterocyte stress response, and failure of key downstream expression of important cytokines and growth factors have been implicated as major factors in intestinal transplant outcomes, namely graft loss and septic death. Treatment strategies with anti tumor necrosis factor (TNF) a, similar to inflammatory bowel disease, have been employed in intestinal transplantation with promising results. Summary In intestinal transplantation, there is evidence that the classical alloimmunity pathways that lead toward graft dysfunction and eventual graft loss may, in fact, be working in concert with a disordered innate immune system to produce a state of chronic inflammation not unlike that seen in inflammatory bowel disease. Keywords inflammatory bowel disease, intestine transplant, NOD2
INTRODUCTION The human intestinal tract exists in a constant state of balance between the destructive capabilities of microbes and the active protective mechanisms of the gut epithelium. Any condition that affects the fine balance of the gut protective mechanisms has the potential to disrupt the chemical and biological immune interfaces of the gut epithelium and produce a disease state. Inflammatory bowel disease (IBD), of which Crohn’s disease and ulcerative colitis are the two most common forms, has origins that are most likely multifactorial in nature [1]. Genetic and environmental factors seem to play an important role, and recent developments have further provided substantial evidence of a dysregulated immune response in a compromised gut epithelium as a major contributor to this disease state [2,3]. A similar state of a compromised gut epithelium is observed after transplantation of the human www.co-transplantation.com
intestine. From the very onset, the gut epithelium is rendered an injury due to the brain death condition of the donor, procurement, and preservation process. Obligate ischemia and reperfusion injury contribute toward epithelial injury, and from then, a lifetime of chronic immunological assault ensues as the host relentlessly attempts allograft destruction, despite chronic immunosuppression. In areas of gut epithelial injury due to all the above factors, a weakened antimicrobial barrier can activate the innate immune response producing a MedStar Georgetown Transplant Institute, Georgetown University Hospital, Washington, DC, USA Correspondence to Cal S. Matsumoto, MD, MedStar Georgetown Transplant Institute, 2 Main, Georgetown University Hospital, 3800 Reservoir Rd., NW, Washington DC, USA. Tel: +1 202 444 3634; fax: +1 202 444 7304; e-mail: [email protected] Curr Opin Organ Transplant 2014, 19:276–280 DOI:10.1097/MOT.0000000000000077 Volume 19 Number 3 June 2014
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Chronic mucosal inflammation after transplantation Matsumoto et al.
KEY POINTS A significant positive association is observed in patients with intestinal failure and the NOD2 genotype mutation, a known risk factor for the development of Crohn’s disease. Recipients of an intestinal transplant with an NOD2 mutation have a higher risk of graft loss and septic death possibly due to a dysregulated innate immune system initiating an injurious inflammatory cascade like what is observed in inflammatory bowel disease. NOD2 mutation intestine transplant recipients have a reduced ability to prevent bacterial adherence, express a greater amount of stress cytokines, and have an impaired Paneth cell antimicrobial response. Intestine transplant recipients with ileal ulcerations have been observed to respond in a similar manner as patients with inflammatory bowel disease to anti-TNF-a therapy, suggesting a similar pathophysiologic mechanism.
scenario similarly observed in nontransplant patients with IBD. In addition, a genetic predisposition with faulty microbial sensoring mechanisms may play a similar role in intestinal transplantation as in IBD.
THE NOD2 POLYMORPHISM AND INFLAMMATORY BOWEL DISEASE-LIKE INJURY AFTER INTESTINAL TRANSPLANT The first description of the potential commonality of a dysregulated innate immune response in the transplanted human intestine and the disease state of IBD was described by Fishbein et al. [4]. In 30 intestinal transplant recipients, an intestinal transplant–IBD phenotype was described in 10% of cases, with gross features of graft ulcerations and a significantly decreased number of Paneth cells, the cells chiefly responsible for the innate immune response, on histological examination. In addition, ulceration healing was noted in 75% of cases with a mean response time of 6 weeks with the administration of anti-tumor necrosis factor (anti-TNF) therapy. Further studies by the same authors led to the discovery, in 2008, of the significant association between NOD2 polymorphisms and poor outcomes after intestinal transplantation [5]. The NOD2 gene encodes for an intracellular protein that serves as a microbial sensor in macrophages, dendritic cells, and certain intestinal epithelial cells, such as Paneth cells. Through these intestinal epithelial cells and gut-associated immune cells, the NOD2 protein promotes the expression of antimicrobial peptides (HD5, HD6), cytokines (TNF-a), and chemokines
(CX3CL1). Mutations of certain several alleles have been identified as functionally abnormal, with an increased risk for Crohn’s disease between two- and 40-fold, depending on the genotype of the individual [6,7]. In Fishbein’s analysis of intestinal transplant recipients with the NOD2 gene mutation, the likelihood of allograft failure was 97-fold higher than recipients with wild-type NOD2 loci. Additionally, in NOD2 mutant recipients, prior to observing an allograft rejection episode, a significant decrease in epithelial antimicrobial peptides human defensin 5 (HD5) and human b-defensin 2 (HBD2) was observed, despite normal bowel function and histology at the time of sampling. The authors conclude that the NOD2 mutant recipient of an intestinal transplant represents a significant risk factor for both rejection and death with the early event of this process involving the impaired expression of epithelium-derived antimicrobial peptides. Further evidence of the association of an NOD2 polymorphism resulting in a dysregulated innate immune system in intestinal failure (IF) patients as well as follow-up of certain subgroups after intestine transplant was reported by Ningappa et al. [8]. In an analysis of 60 Caucasian children with a primary intestine transplant, Ningappa specifically identified the NOD2 mutation at the R702W allele as a significant risk allele for the need of a combined liverintestine transplant as compared with 538 healthy controls of the same racial background (13.5 versus 3.6%, P ¼ 0.0007). The R702W allele was further identified as a significant risk allele associated with early rejection episodes (P ¼ 0.028) independent of other covariates in a linear regression analysis. Patient survival after liver-intestine transplant in those with the R702W allele was also significantly decreased (P ¼ 0.015) with half of the deaths resulting from septic events. The strong association of these NOD2 mutant IF patients requiring a liver-inclusive intestine transplant is best explained by the defect in the epithelial innate immune system contributing to a chronic epithelial bacterial invasion and portal endotoxemia which eventually leads to liver dysfunction and liver failure. In the posttransplant setting, the poor outcome associated with a recipient NOD2 polymorphism may represent a continuing susceptibility to inflammatory damage (which is an intrinsic characteristic of the recipient) or may be due to the potentiating effects of the inflammation on the graft alloimmunity process.
THE NOD2 POLYMORPHISM AND ASSOCIATION WITH INTESTINAL FAILURE Two recent studies have also highlighted the contribution of the NOD2 mutation may have on IF
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Small bowel transplantation &
itself. Schaffler et al. [9 ] genotyped 46 non-Crohn’s disease patients with IF and observed a significant increase in the frequency of the NOD2 allele mutation as compared with healthy controls (14 versus 6%, P ¼ 0.006). Despite the higher incidence of IF with the NOD2 mutation, this did not translate into an increased need for intestinal transplantation in their analysis, most likely due to the overall low incidence of intestinal transplants performed at their center. In an extension of the original analysis by Fishbein, Guerra et al. [10 ] analyzed the NOD2 allele frequency of 192 consecutive patients with IF as compared with 103 healthy controls. Overall incidence of NOD2 mutation in the IF group was significantly greater than healthy controls (26 versus 4.8%, P < 0.0001) and even with the exclusion of Crohn’s disease patients, the positive influence of NOD2 mutation status remained significant (18.8 versus 4.8%, P ¼ 0.001). &
EPITHELIAL CELL DYSREGULATION IN INFLAMMATORY BOWEL DISEASE AND AFTER INTESTINAL TRANSPLANT An association between intestinal epithelial cell barrier dysfunction and IBD has been well established in several independent studies. A critical factor in the pathogenesis of IBD is the disturbed antimicrobial defense provided by the intestine innate immune system. A conspicuous lack of Paneth cell a-defensins in ileal Crohn’s disease , both with the wild-type NOD2 phenotype and, more pronounced, in the NOD2 mutant phenotype has been established as a key element linking a dysfunctional intestinal innate immune system to IBD [11–14]. In Fishbein’s original work, a reduced expression of HD5 was observed in intestinal transplant recipients with the NOD2 mutation. Lough et al. [15 ] has further defined this work by observing not only reduced antimicrobial expression in NOD2 mutation–engrafted intestinal transplant recipients, but also a reduced ability to prevent bacterial adherence, a significant increased expression of CX3CL1, a stress cytokine produced by the injured enterocyte, and a failure of the NOD2 mutant intestinal transplant recipient to express key genes, such as Wnt5a, which regulate Paneth cell activation. Twenty-four non-Crohn’s disease recipients of an intestine transplant were evaluated, 12 with the NOD2 mutation and 12 with the wildtype NOD2 genotype. Bacterial adherence was examined with a probe (EUB338) targeting bacterial 16S rRNA by fluorescent in-situ hybridization methods. Consistent observations of relatively bacteria-free epithelial adherence was noted in the NOD2 wild-type recipients as compared with the NOD2 mutant recipients by averaged fluorescent intensity &&
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(P 0.05), leading to the speculation that this results as a consequence of the failure of Paneth cell function to produce effective levels of antimicrobial peptides in NOD2 mutant recipients. Further evidence of the downstream dysregulation of the epithelium in the NOD2 mutant recipients was also addressed by the authors. The cytokine CX3CL1, a recognized injury-related protein, was observed by semiquantitative analysis as well as by relative mRNA reverse transcription PCR to be significantly more expressed in the NOD2 mutant recipients, indicating a greater epithelial stress response, most likely from the overabundance of adherent pathogenic microbes, as described above. The most striking observation made in this report was the discovery that a myeloid cell of recipient origin, and which carried an NOD2 mutation appeared to be functionally defective. Unlike the comparable cell from NOD2 wild-type recipients, this cell failed to extend processes into the surface epithelial layer, suggesting it was no longer capable of monitoring the apical microenvironment. The NOD2 mutant myeloid cell also failed to express many cytokines and growth factors secreted by the NOD2 wild-type cell. These included Wnt5a, a growth factor known to stimulate expression of antimicrobial peptides and proteins, including HD5, within the Paneth cell. In NOD2 mutant recipients, lower secretions of Wnt5a as well as Wnt5a mRNA expression were observed as compared with NOD2 wild type (P 0.05). The finding that Wnt5a is secreted by a ‘sentinel’ myeloid cell within the intestinal lamina propria suggests that these cells coordinate with Paneth cell antimicrobial activity to match the threat of microbial invasion at the epithelial boundary. In addition, it is conceivable that this same ‘sentinel’ cell also plays a role in renewal rate of the small intestinal epithelium, since Wnt5a also stimulates the rate of stem cell cycling within the intestinal crypt. Furthermore, through its secretion of numerous cytokines, the same cell might also help orchestrate the community of immune cells within the lamina propria to effectively manage microbial invasion into that compartment. This evidence, from comparable intestine transplant recipients with differing NOD2 genotype status, may provide a provocative explanation to account for the increased morbidity and mortality associated with intestinal transplantation in general.
USE OF ANTI-TNF AGENTS IN INTESTINAL TRANSPLANT Perhaps the most compelling clinical evidence of the similar association between IBD and the Volume 19 Number 3 June 2014
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Chronic mucosal inflammation after transplantation Matsumoto et al.
intestinal transplant recipient comes in the form of the clinical response of these patients from the administration of TNF-a inhibitors. TNF-a has been firmly established as playing a pivotal role in the inflammatory response that is orchestrated in patients suffering from IBD. Through its proinflammatory signaling pathways, TNF-a upregulates other inflammatory cytokines and, among other nefarious activities, it induces epithelial cell apoptosis as well as a degradation of epithelial expression of tight junctions, further causing exacerbation of inflammation through a weakened mucosal barrier [16]. In patients with IBD, concentrations of TNF-a are elevated in the blood, intestinal mucosa, and stool [17]. TNF-a inhibitors, not surprisingly, have significantly altered the treatment landscape of IBD in the last decade with superior remission rates and improved mucosal healing in IBD than what had been observed with conventional medical therapy [18]. As reported by Pascher et al. with the successful treatment of two intestinal transplant recipients with Orthoclone (OKT3)-resistant ulcerative inflammation and similarly observed by Fishbein in 2006 with the healing of intestinal transplant ulceration in 75% of the cases, the use of anti-TNF-a agents and the similar outcomes as with IBD patients may imply that similar forces are behind the molecular pathophysiology of transplant graft ulceration and dysfunction [4,19]. The specific role in intestinal transplantation, however, remains less clear. Gerlach et al. [20] reported the use of Infliximab (Remicaide; Janssen Biotech (formerly Centocor Inc.), Horsham, PA, USA; ESSEX PHARMA GmbH, Munich, Germany) in nine intestine transplant patients: two with early onset OKT3-resistant acute cellular rejection (ACR) two with ulcerative distal graft inflammation, and five with late onset steroid-resistant ACR or OKT3-resistant ACR with chronic ulcerative graft changes. In eight of nine patients, Infliximab administration resulted in resolution of elevated serum TNF-a levels together with resolution of graft changes of either a chronic ulcerative nature or acute cellular injury. One patient who did not respond had most likely a significant humoral component injury; of which Th1-driven inflammatory antagonist may not play a role. Most striking was the reproducible success of Infliximab with late onset graft ulceration, which represents a more similar clinical setting; of which the successful use of Infliximab in IBD has been observed. A more recent report from De Greef et al. [21] describes the use of Infliximab for the treatment of refractory ACR in two pediatric intestinal transplant recipients. No chronic graft ulcerations were noted in these cases, and infliximab was used as a salvage therapy for
thymoglobulin-resistant rejection, possibly implicating TNF-a as a major mediator for ACR, as similarly observed by Gerlach. With limited data from the intestinal transplant experience, however, we can only postulate that the role played by antiTNF-a therapy has similar mechanisms to that of in IBD patients.
CONCLUSION The current extent of our knowledge regarding the actual pathophysiology of intestine allograft rejection remains limited. From an epidemiological standpoint, there is strong evidence that patients with IF itself may share certain immutable traits as with those suffering from IBD. Evidence from the field of IBD points to a dysregulation of the innate immune system, whether inherent or acquired, as the chief mediator of IBD pathology. In intestinal transplantation, there is evidence that the classical alloimmunity pathways that lead toward graft dysfunction and eventual graft loss may, in fact, be working in concert with a disordered innate immune system to produce a state of chronic inflammation not unlike that seen in IBD. Given the intricate immunologic and environmental variables inherent and unique to the human intestine, there may possibly be a multitude of pathways that lead to graft destruction and loss. Whether it is a disordered innate immune system that is a product of a classic human leukocyte antigen host–graft immunologic interaction or simply an inherent quality of certain high-risk recipient genotypes is a question that needs an answer with further research. The truth for intestinal transplantation most likely lies in-between as a combination that has been described previously as the third form of IBD. Acknowledgements None. Conflicts of interest There are no conflicts of interest.
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Volume 19 Number 3 June 2014
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