Scandinavian Journal of Gastroenterology. 2014; 49: 157–163
ORIGINAL ARTICLE
Effect of inflammatory bowel disease therapies on immunogenicity of Mycobacterium paratuberculosis proteins
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ANNE XIA1,*, JOANNE M. STEMPAK2,*, JESSE GRIST3, BRIAN BRESSLER4, MARK S. SILVERBERG2 & HORACIO BACH1 1
Division of Infectious Diseases, Department of Medicine, University of British Columbia, Vancouver, BC, Canada, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada, 3Earl Marriott Secondary School, Surrey, BC, Canada, and 4Division of Gastroenterology, Department of Medicine, University of British Columbia, Vancouver, BC, Canada 2
Abstract Background and aims. The link between Mycobacterium avium subsp. paratuberculosis (MAP) and Crohn’s disease (CD) is supported by several studies that have reported the detection and isolation of MAP from human tissues, but causation has not yet been proven. Preliminary studies have shown higher levels of antibodies in sera from CD patients against secreted protein from MAP within human macrophages when compared to healthy controls. The immunogenicity of this protein in CD patients under different treatment regimes was evaluated. Materials and methods. Sera of 110 CD patients, 82 ulcerative colitis (UC), and 150 healthy controls were collected and the presence of antibodies against the mycobacterial protein tyrosine phosphatase PtpA was assayed using ELISA. Results. A statistically significant difference in the level of antibodies against PtpA was measured in untreated CD patients versus healthy controls, but variation in the antibody levels was observed when patients were subjected to different treatment regimens. UC patients showed no differences in the levels of antibodies against PtpA when compared to healthy controls. Conclusions. CD patients under different drug treatments show a clear difference in the levels of antibodies against a protein secreted by MAP, suggesting that if MAP is active in the progress of CD, some treatments can be detrimental to its survival.
Key Words: Crohn’s disease, Mycobacterium avium ssp. paratuberculosis, signal transduction, survival, therapies, ulcerative colitis
Introduction Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease (JD); a chronic, debilitating, and fatal enteritis in livestock and wild animals with symptoms similar to those observed in Crohn’s disease (CD) [1]. Several studies link an association between MAP and CD [2,3], although this connection is still under study. Interestingly, MAP has been detected and isolated from human tissues [4–6] including serum [7,8], and body fluids such as breast milk [9]. Moreover, the
presence of MAP in dairy products suggests a potential vehicle for acquisition of this pathogen by humans [10,11], especially since it is resistant to pasteurization [12]. Interestingly, higher levels of TNF-a are secreted by gut mucosa in MAP-associated CD patients [13]. Recently, an increase in the secretion of TNF-a from human macrophages infected with MAP, but not with M. avium or M. smegmatis has also been reported [14]. In addition, when exposed to infliximab, an antiTNF-a antibody, the survival of MAP in infected macrophages decreased [15], supporting the premise
Correspondence: Horacio Bach, Division of Infectious Diseases, Department of Medicine, University of British Columbia, Jack Bell Research Centre, 410-2660 Oak Street, Vancouver, British Columbia, V6H 3Z6, Canada. Tel: +1 604 875 4111. Tel: x 62107. Fax: +1 604 875 4013. E-mail: [email protected] *Both authors contributed equally to this work.
(Received 7 September 2013; revised 13 October 2013; accepted 16 October 2013) ISSN 0036-5521 print/ISSN 1502-7708 online 2014 Informa Healthcare DOI: 10.3109/00365521.2013.857713
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that this pathogen is able to modulate the immune system for its own benefit. Macrophages engulf microorganisms by enclosing them in vesicles termed phagosomes, which fuse with lysosomes, organelles in charge of vesicle cargo digestion including phagosomes. MAP is able to infect, reside, and multiply in bovine and human macrophages [16–18], suggesting that the pathogen is able to elude the host’s immune response. Although the exact survival mechanism of MAP in macrophages remains to be elucidated, our previous report showed the secretion of the mycobacterial protein tyrosine phosphatase (PtpA) in a time-dependent manner on MAP infection of human-derived monocyte cell line THP-1 [19]. The function of PtpA has been elucidated in M. tuberculosis, a very similar pathogen, which also secretes PtpA and shares a 90% homology to MAP PtpA [18]. PtpA inhibited phagosome acidification [19] and the phagosome–lysosome fusion event in human macrophages by dephosphorylating the host sorting protein VPS33B [20]. By avoiding the lysosome, the pathogen escapes host immunological surveillance and establishes successful infection. We hypothesized that as in the case of M. tuberculosis, PtpA also promotes the survival of MAP in host cells, and as a secreted protein within macrophages it would be processed as foreign protein, and presented to antibody-producing cells. Then, if MAP is able to infect humans, we would expect that antibodies against PtpA are present in serum of CD patients as a result of antigen processing. Indeed, recently we found significantly higher levels of antibodies in CD patients against PtpA when compared to healthy controls [17], and as mentioned above, a decrease in the levels of antibodies against PtpA was measured in CD patients under infliximab treatment [15]. Following our previous studies, we analyzed antibody levels against PtpA in a bigger cohort of CD patients and examined the variation of these antibodies depending on the treatment provided. In addition, levels of antibodies against PtpA were measured in ulcerative colitis (UC) patients. Methods Patients The research ethics board of Mount Sinai Hospital, Toronto, Ontario, Canada, approved the protocol for this study. All subjects were recruited at Mount Sinai Hospital in Toronto, Canada between the years of 2003 and 2011 and provided written informed consent prior to sample collection. Serum samples were obtained from peripheral blood from 110 patients with a proven diagnosis of CD, 82 patients diagnosed
with UC, and 150 patients without CD or UC (healthy controls). The inclusion criteria for subjects are as follows: i) No previous history of tuberculosis infection, ii) No major risk factors for tuberculosis (HIV positive status, organ transplant recipient, known contact with TB patients, and iii) No antibiotic use which may potentially have activity against Mycobacteria in the previous 6 months. Information regarding demographic, medication history, and previous disease course were collected at study entry. Based on the clinical records obtained to perform this study, we were not able to stratify the two forms of CD (perforating and non-perforating) and the data show in this study is presented as a “single” form of CD [21]. Blood collection Peripheral blood collected from CD patients, UC patients, and controls was processed in order to obtain sera. Whole blood (5–10 ml) was collected in tubes without any additives at Mount Sinai Hospital. Each specimen was labeled with a study code and the serum was obtained after centrifugation of the coagulated whole blood at 3000 rpm for 10 min. Serum was aliquoted in cryovials with external caps and internal O-ring seals and kept at 80oC. The investigators performing the ELISA tests were blinded to the clinical characteristics associated with each specimen. Bacterial strains MAP strains K-10 (ATCC BAA-968) was cultured in Middlebrook 7H9 broth (BD) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (BD), 0.05% Tween-80 (Fisher), and 2 g/l mycobactin J (Allied Monitor). When necessary, solid medium was prepared by adding 1.5% agar (BD). MAP was cultured in rolling bottles and 0.7 108 cells were inoculated in the broth. Cultures were exposed to different concentrations of drugs currently used for CD treatments. Prednisone (Sigma-Aldrich) was dissolved in DMSO and used at final concentrations of 12.5, 62.5, 1,250 mg/ml; whereas 5-aminosalycilic acid (5-ASA) also dissolved in DMSO was used at final concentrations of 10, 25, 50, and 100 mg/ml. Bacterial growth was monitored at OD of 600 nm. Antigen Production and ELISA Recombinant PtpA was produced in M. smegmatis and according to published procedures [18]. Ninty-six-well flat bottom plates were coated with 50 mg of recombinant PtpA in 50 ml of coating buffer (0.1 M NaHCO3 pH 9.6) and placed at 4 C overnight. Next day, wells were washed (3) with phosphate buffer saline (PBS),
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Variation of MAP proteins immunogenicity in IBD patients and blocked with 200 ml of 3% bovine serum albumin (BSA) in PBS overnight at 4 C. A serum dilution of 1:100 was found to be the optimal dilution. Thus, sera were diluted 1:100 in 3% BSA, and 50 ml was added to each well. After 2 h incubation at room temperature, wells were washed (3) with PBS supplemented with 0.05% Tween-20 (PBS-T). Goat anti-human coupled to horseradish peroxidase (Jackson) was used as secondary antibody diluted 1:5000 in 3% BSA solution. After 1 h exposure, wells were washed (3) with PBS-T and 50 ml of a solution of 3,3¢,5,5¢-tetramethylbenzidine (Alfa Aesar) was used as the developer. Reactions were stopped with a solution of 25 ml of 1 M sulfuric acid. Plates were read at 450 nm using an ELISA reader. Fetal calf serum and the secondary antibody were used as a negative control. All the experiments were performed in triplicate. Statistical analysis To compare ELISA tests between CD and UC positive and healthy patients for PtpA, assuming a nonnormal distribution, Wilcoxon rank sum test was used and a Bonferroni correction for multiple comparisons was implemented. p < 0.05 was considered statistically significant. Results Antibodies against PtpA in sera of CD and UC patients A total of 342 archived samples corresponding to 110 CD, 82 UC patients, and 150 controls were
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analyzed in this study. All CD and UC patients had well-characterized disease based on clinical, endoscopic, radiologic, and histological criteria (Table I). When sera of CD and UC patients and controls were screened for antibodies against PtpA, a mean antibody intensity reading of 0.232 and 0.145 were calculated for CD and UC patients, respectively, whereas a mean of 0.108 was measured in the control group (Figures 1 and 2). CD (p < 0.0001) but not UC patient(p = 0.3124) sera showed significantly higher titer of antibodies against PtpA when compared to controls.
Effect of CD and UC treatments on the level of antibodies against PtpA The impact of usual therapies typically taken by patients with IBD include azathioprine (AZA), 5-ASA, infliximab, and steroids including both prednisone and budesonide. We were interested in determining whether treatments with these medications alter levels of antibodies against PtpA, because our previous studies found that CD patients treated with infliximab had significantly decreased levels of PtpA antibodies. Variability in the level of antibodies against PtpA was observed when sera from treated patients were analyzed. A statistical difference was observed between CD patients treated with AZA and untreated CD patients, whereas no significant difference was observed when patients were treated with either 5-ASA or steroids (Figure 2). Interestingly, when AZA was combined with other medications, such as 5-ASA and/or steroids, no significant difference was measured (Figure 2).
Table I. Clinical characteristics of study patients (n = 342).
Age in year, median (range) Gender, female n (%) Years of disease, median ± SD Medical therapy, n (%) Prednisone Azathioprine Methotrexate Budesonide 5-ASA Combined* None Previous surgeries, n (%) Perianal fistula Colostomy Hemi colostomy Ileal resection Ileocolonic resection Small bowel (other than ileum) resection
CD patients (n = 110)
UC patients (n = 82)
Healthy controls (n = 150)
32 (17–81) 51 (46) 8.5 ± 8.8
34 (23–53) 38 (46) 8 ± 7.8
38 (17–85) 79 (53) n/a
8 (10) n/a n/a 1 (1) 34 (41) 21 (26) 18 (22)
n/a n/a n/a n/a n/a n/a n/a
n/a n/a n/a n/a n/a n/a
n/a n/a n/a n/a n/a n/a
6 16 3 9 8 31 37
(5) (14) (3) (8) (7) (28) (33)
1 (0.9) 1 (0.9) 4 (4) 28 (25) 1 (0.9) 3 (3)
Abbreviation: n/a = non applicable. *Combined, a combination with at least two of the listed drugs.
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Figure 1. Distribution of the antibody intensity readings. A boxplot analysis shows the distribution of the antibody intensity readings obtained by ELISA in untreated CD and UC patients. *p-Values of 0.0009 and < 0.001 were obtained using Wilcoxon rank sum test and Bonferroni test, respectively. OD = optical density; HC = healthy controls; CD = Crohn’s disease; UC = Ulcerative colitis. Experiments were performed in triplicate.
In the case of UC patients, no differences in PtpA antibody levels were observed when patients were treated with AZA, 5-ASA, and steroids, singly or combined (Figure 3). Exposure of MAP to 5-ASA and prednisone Previous studies reported impaired growth of MAP when it was cultured in the presence of AZA [22] or infliximab [15], suggesting that exposure of MAP to these drugs affects the normal physiology of the pathogen in vitro. To determine the effect of 5-ASA and steroids on MAP growth, the pathogen was exposed to different concentrations of both compounds in vitro. In both cases, an increase in MAP proliferation was recorded, indicating that these compounds have no detrimental effect in the normal growth of MAP (Figure 4A, B). In the case of 5-ASA, MAP growth was intensified with exposed to a concentration of >25 mg/ml when compared to the non-treated control (Figure 4A).
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Figure 2. Distribution of the antibody intensity readings in CD untreated and treated patients. A boxplot analysis shows the distribution of the antibody intensity readings obtained by ELISA in untreated CD patients, CD patients under individual treatment with azathioprine (AZA) (A), 5-ASA (M), and steroids (S) or their combinations (A+M+S). *p-Values of 0.0032 < 0.001 were obtained using Wilcoxon rank sum test and Bonferroni test, respectively. OD = optical density; CD = Crohn’s disease. Experiments were performed in triplicate.
CD patients in comparison to controls in small cohorts [15,17]. Moreover, levels of antibodies in CD patients treated with infliximab showed no statistical differences when compared to controls,
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Discussion To explore whether current treatments for CD cause variability in the level of antibodies against PtpA, pilot analysis was conducted on archived serum samples to evaluate the presence of these antibodies. Sera of untreated UC patients, as well as UC patients under different treatments, were also tested. We have shown in two previous studies that levels of antibodies against PtpA were significantly higher in
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Figure 3. Distribution of the antibody intensity readings in UC untreated and treated patients. A boxplot analysis shows the distribution of the antibody intensity readings obtained by ELISA in untreated UC patients, UC patients under individual treatment with 5-ASA (M), and steroids (S) or their combinations (A+M+S). OD = optical density; UC = ulcerative colitis. Experiments were performed in triplicate.
Variation of MAP proteins immunogenicity in IBD patients B
OD (l = 600 nm)
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Control 66 µg/ml 166 µg/ml 333 µg/ml 666 µg/ml
1.0 0.8 0.6 0.4 0.2
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Control 12.5 µg/ml 62.5 µg/ml 1250 µg/ml
1.0 OD (l = 600 nm)
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90 120 Time (h)
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Figure 4. Growth curve of MAP exposed to (A) 5-ASA and (B) prednisone. MAP cultures were exposed to increasing concentrations of 5-ASA and prednisone and sampled at specific time points. The optical density of the culture was measured at 600 nm. Shown are the mean values (±SE) of three independent experiments.
suggesting that inhibition of TNF-a was detrimental either to the survival of the pathogen or to the establishment of a successful infection, or that infliximab treatment affects the antigen presentation system of macrophages. In this study, a significant difference in the levels of antibodies against PtpA in CD-treated versus nontreated patients was observed (Figure 1). These results are in concord with our preliminary study performed in a smaller cohort [17] and reinforce the role this novel antibody can play in assessing patient’s exposure to MAP. Interestingly, variability in the levels of the antibodies against PtpA was observed when CD patients were subjected to different treatments. For instance, in this study, sera from CD patients treated with AZA had decreased levels of antibodies; while sera from 5-ASA or prednisonetreated CD patients did not show any significant difference in antibody levels. Studies on the effects of different non-antibiotic drugs, such as 5-ASA [23], AZA [22], cyclosporine A [24], methotrexate [25], rapamycin [24], tacrolimus [24], and thalidomide [26] on MAP growth have been reported. The immunosuppressant AZA appears to inhibit MAP growth in vitro [22] and in our study, a decrease in the level of antibodies against PtpA was measured in CD patients treated with this drug. This finding suggests that if AZA inhibits the proliferation of MAP, a concomitant decrease in the levels of antibodies against PtpA would be observed due to the reduction of MAP cells. On the other hand, patients treated with 5-ASA or steroids showed no significant differences in antibody levels when compared to untreated CD patients, suggesting that if MAP is active in CD patients, these treatments have no effect on the pathogen. Interestingly, although the use of AZA decreases the levels of antibodies against PtpA, when it is combined with other drugs, these effects are attenuated as each individual drug in the combination may
have a different impact in the regulation of signal transduction pathways. For example, it has been reported that 5-ASA possesses scavenging and antioxidant effects against reactive oxygen and nitrogen species [27]. In addition, 5-ASA activates the production of heme-oxygenase-1, which converts heme into biliverdin releasing carbon monoxide (CO). This particular CO has anti-inflammatory properties including down regulation of the expression of inflammatory cytokines [28,29]. Taking together, the presence of 5-ASA in the drug combination would provide a benefit to MAP because of its anti-inflammatory, antioxidant, and scavenging properties. In the case of 5-ASA, a previous study showed that inhibition of MAP growth was observed when bacilli were exposed to 5-ASA in vitro [23]. The results presented here related to the level of antibodies provide contradictory results because no effects on the antibody levels were observed in patient sera treated with 5-ASA. Differences in the results can be explained by either a difference in MAP species being used or the special cell morphology (spheroplast-like cells) of MAP when it is isolated from human tissues. Since spheroplast MAP cells lack a cell wall, a remodeling of the bacterial surface is expected and drugs can be beneficial or detrimental for MAP growth. Therefore, inhibition of MAP by 5-ASA in vivo would be different when compared to in vitro conditions when the whole cell wall is present [30]. No previous studies have examined the effect of steroids in MAP. In this report, we show that the steroid prednisone does not affect MAP growth. A previous report describing the proliferation of MAP in hamsters treated with the steroid dexamethasone showed significantly higher bacterial counts in spleen and Peyer’s patches [31]. In addition, Mycobacteria are able to degrade cholesterol [32] and the pathogenic M. tuberculosis is able to use cholesterol as a unique carbon source [33]. Since, steroids used for treatments are derivatives of cholesterol and taking in account that
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MAP possesses all the enzymes to degrade cholesterol, steroids can be exploited by the pathogen as a carbon source and that can explain why a higher number of MAP colonies was observed in hamsters treated with the steroid dexamethasone. Results reported here show that significant levels of antibodies against PtpA were not measured in untreated UC patients, suggesting that MAP is not involved in UC etiology. This conclusion, although controversial, is supported by several reports that showed differential findings of MAP in CD and UC patients. For example, a meta-analysis study performed to assess the association of MAP with CD found an association with MAP in CD, but not in UC [34]. Another study reported the amplification of the DNA fragment IS900, commonly used for the specific detection of MAP, in 52% tissue samples from CD patients and only 2% in specimens obtained from UC patients [4]. On the other hand, some studies have reported inconclusive results as no differences in antibodies against MAP antigens were observed among the study groups including CD and UC patients, healthy controls, and non-affected siblings [35]. However, 15% of 29 UC patients reacted positively with a MAP antigen [36] and MAP DNA was detected in 20% of UC patients, but in 33% of healthy controls, suggesting that MAP is widely distributed in the environment including in the food chain [37]. Interestingly, a study performed in Manitoba, Canada found no differences in the association between MAP and healthy controls, CD or UC patients [38]. Results of this study were performed by adapting an ELISA test used by veterinarians to diagnose JD. One explanation for this difference is the changes that MAP undergoes when infecting the human host. Physiological changes render this pathogen devoid of the cell wall, probably as a way to escape from the immune system [3]. Thus, fewer antigens will be available to prime the immune system and fewer antibodies will be measured. A second possible explanation is the fact that the province of Manitoba possesses high incidence and prevalence rates of CD [39]. Thus, the presence of antibodies can be related to an exposure to the pathogen rather than an actual infection. In our case, we measured the antibodies against a virulence factor of the pathogen, which is secreted in a constant manner only within the host. Our results also concord with other studies where differences between CD patients and healthy controls were found using the MAP antigens p35 and p36 [36]. In conclusion, we found different levels of PtpA antibodies against MAP in human sera, which vary according to the medical treatment of CD patients, but no significant differences were observed in UC
patients, suggesting that MAP is not associated with this disease. Serial measurements of PtpA antibodies in patients with CD treated with various medications may be another outcome that should be followed to optimize medical care. Further research will be done to understand this novel approach to CD treatment.
Acknowledgement We are very thankful to Jeffrey Helm for helpful discussion. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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Variation of MAP proteins immunogenicity in IBD patients [12] Gao A, Mutharia L, Chen S, Rahn K, Odumeru J. Effect of pasteurization on survival of Mycobacterium paratuberculosis in milk. J Dairy Sci 2002;85:3198–205. [13] Clancy R, Ren Z, Turton J, Pang G, Wettstein A. Molecular evidence for Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn’s disease correlates with enhanced TNFalpha secretion. Dig Liver Dis 2007;39:445–51. [14] Nakase H, Tamaki H, Matsuura M, Chiba T, Okazaki K. Involvement of Mycobacterium avium subspecies paratuberculosis in TNF-alpha production from macrophage: possible link between MAP and immune response in Crohn’s disease. Inflamm Bowel Dis 2011;17:E140–2. [15] Bach H, Rosenfeld G, Bressler B. Treatment of Crohn’s disease patients with infliximab is detrimental for the survival of Mycobacterium avium ssp. paratuberculosis within macrophages and shows a remarkable decrease in the immunogenicity of mycobacterial proteins. J Crohns Colitis 2012;6: 628–9. [16] Bannantine JP, Stabel JR. Killing of Mycobacterium avium subspecies paratuberculosis within macrophages. BMC Microbiol 2002;2:2. [17] Bach H, Ko HH, Raizman EA, Attarian R, Cho B, Biet F, et al. Immunogenicity of Mycobacterium avium subsp. paratuberculosis proteins in Crohn’s disease patients. Scand J Gastroenterol 2011;46:30–9. [18] Bach H, Sun J, Hmama Z, Av-Gay Y. Mycobacterium avium subsp. paratuberculosis PtpA is an endogenous tyrosine phosphatase secreted during infection. Infect Immun 2006;74:6540–6. [19] Wong D, Bach H, Sun J, Hmama Z, Av-Gay Y. Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+-ATPase to inhibit phagosome acidification. Proc Natl Acad Sci USA 2011;108:19371–6. [20] Bach H, Papavinasasundaram KG, Wong D, Hmama Z, Av-Gay Y. Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B. Cell Host Microbe 2008;3:316–22. [21] Greenstein AJ, Lachman P, Sachar DB, Springhorn J, Heimann T, Janowitz HD, et al. Perforating and nonperforating indications for repeated operations in Crohn’s disease: evidence for two clinical forms. Gut 1988;29:588–92. [22] Shin SJ, Collins MT. Thiopurine drugs azathioprine and 6mercaptopurine inhibit Mycobacterium paratuberculosis growth in vitro. Antimicrob Agents Chemother 2008;52: 418–26. [23] Greenstein RJ, Su L, Shahidi A, Brown ST. On the action of 5-amino-salicylic acid and sulfapyridine on M. avium including subspecies paratuberculosis. PLoS ONE 2007;2:e516. [24] Greenstein RJ, Su L, Juste RA, Brown ST. On the action of cyclosporine A, rapamycin and tacrolimus on M. avium including subspecies paratuberculosis. PLoS ONE 2008;3: e2496. [25] Greenstein RJ, Su L, Haroutunian V, Shahidi A, Brown ST. On the action of methotrexate and 6-mercaptopurine on M. avium subspecies paratuberculosis. PLoS ONE 2007;2:e161. [26] Greenstein RJ, Su L, Brown ST. On the effect of thalidomide on Mycobacterium avium subspecies paratuberculosis in culture. Int J Inf Dis 2009;13:e254–63.
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[27] Ahnfelt-Rønne I, Nielsen OH, Christensen A, Langholz E, Binder V, Riis P. Clinical evidence supporting the radical scavenger mechanism of 5-aminosalicylic acid. Gastroenterology 1990;98:1162–9. [28] Freitas A, Alves-Filho JC, Secco DD, Neto AF, Ferreira SH, Barja-Fidalgo C, et al. Heme oxygenase/carbon monoxidebiliverdin pathway down regulates neutrophil rolling, adhesion and migration in acute inflammation. Br J Pharmacol 2006;149:345–54. [29] . Otterbein LE, Bach FH, Alam J, Soares M, Lu HT, Wysk M, et al.et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 2000;6:422–8. [30] Wall S, Kunze ZM, Saboor S, Soufleri I, Seechurn P, Chiodini R, et al. Identification of spheroplast-like agents isolated from tissues of patients with Crohn’s disease and control tissues by polymerase chain reaction. J Clin Microbiol 1993;31:1241–5. [31] Larsen AB, Miller JM. Effect of dexamethasone on Mycobacterium paratuberculosis infection in hamsters. Am J Vet Res 1978;39:1866–7. [32] Van der Geize R, Yam K, Heuser T, Wilbrink MH, Hara H, Anderton MC, et al. A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci USA 2007;104:1947–52. [33] Thomas ST, VanderVen BC, Sherman DR, Russell DG, Sampson NS. Pathway profiling in Mycobacterium tuberculosis: elucidation of cholesterol-derived catabolite and enzymes that catalyze its metabolism. J Biol Chem 2011; 286:43668–78. [34] Feller M, Huwiler K, Stephan R, Altpeter E, Shang A, Furrer H, et al. Mycobacterium avium subspecies paratuberculosis and Crohn’s disease: a systematic review and meta-analysis. Lancet Infect Dis 2007;7:607–13. [35] Bernstein CN, Blanchard JF, Rawsthorne P, Collins MT. Population-based case control study of seroprevalence of Mycobacterium paratuberculosis in patients with Crohn’s disease and ulcerative colitis. J Clin Microbiol 2004;42: 1129–35. [36] Naser SA, Hulten K, Shafran I, Graham DY, El-Zaatari FA. Specific seroreactivity of Crohn’s disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis. Vet Microbiol 2000;77:497–504. [37] Rath T, Roderfeld M, Blocher S, Rhode A, Basler T, Akineden O, et al. Presence of intestinal Mycobacterium avium subspecies paratuberculosis (MAP) DNA is not associated with altered MMP expression in ulcerative colitis. BMC Gastroenterol 2011;11:34. [38] Bernstein CN, Blanchard JF, Rawsthorne P, Collins MT. Population-based case control study of seroprevalence of Mycobacterium paratuberculosis in patients with Crohn’s disease and ulcerative colitis. J Clin Microbiol 2004;42: 1129–35. [39] Bernstein CN, Blanchard JF, Rawsthorne P, Wadja A. Epidemiology of Crohn’s disease and ulcerative colitis in a central Canadian province: a population-based study. Am J Epidemiol 1999;149:916–24.
ORIGINAL ARTICLE
Effect of inflammatory bowel disease therapies on immunogenicity of Mycobacterium paratuberculosis proteins
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ANNE XIA1,*, JOANNE M. STEMPAK2,*, JESSE GRIST3, BRIAN BRESSLER4, MARK S. SILVERBERG2 & HORACIO BACH1 1
Division of Infectious Diseases, Department of Medicine, University of British Columbia, Vancouver, BC, Canada, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada, 3Earl Marriott Secondary School, Surrey, BC, Canada, and 4Division of Gastroenterology, Department of Medicine, University of British Columbia, Vancouver, BC, Canada 2
Abstract Background and aims. The link between Mycobacterium avium subsp. paratuberculosis (MAP) and Crohn’s disease (CD) is supported by several studies that have reported the detection and isolation of MAP from human tissues, but causation has not yet been proven. Preliminary studies have shown higher levels of antibodies in sera from CD patients against secreted protein from MAP within human macrophages when compared to healthy controls. The immunogenicity of this protein in CD patients under different treatment regimes was evaluated. Materials and methods. Sera of 110 CD patients, 82 ulcerative colitis (UC), and 150 healthy controls were collected and the presence of antibodies against the mycobacterial protein tyrosine phosphatase PtpA was assayed using ELISA. Results. A statistically significant difference in the level of antibodies against PtpA was measured in untreated CD patients versus healthy controls, but variation in the antibody levels was observed when patients were subjected to different treatment regimens. UC patients showed no differences in the levels of antibodies against PtpA when compared to healthy controls. Conclusions. CD patients under different drug treatments show a clear difference in the levels of antibodies against a protein secreted by MAP, suggesting that if MAP is active in the progress of CD, some treatments can be detrimental to its survival.
Key Words: Crohn’s disease, Mycobacterium avium ssp. paratuberculosis, signal transduction, survival, therapies, ulcerative colitis
Introduction Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease (JD); a chronic, debilitating, and fatal enteritis in livestock and wild animals with symptoms similar to those observed in Crohn’s disease (CD) [1]. Several studies link an association between MAP and CD [2,3], although this connection is still under study. Interestingly, MAP has been detected and isolated from human tissues [4–6] including serum [7,8], and body fluids such as breast milk [9]. Moreover, the
presence of MAP in dairy products suggests a potential vehicle for acquisition of this pathogen by humans [10,11], especially since it is resistant to pasteurization [12]. Interestingly, higher levels of TNF-a are secreted by gut mucosa in MAP-associated CD patients [13]. Recently, an increase in the secretion of TNF-a from human macrophages infected with MAP, but not with M. avium or M. smegmatis has also been reported [14]. In addition, when exposed to infliximab, an antiTNF-a antibody, the survival of MAP in infected macrophages decreased [15], supporting the premise
Correspondence: Horacio Bach, Division of Infectious Diseases, Department of Medicine, University of British Columbia, Jack Bell Research Centre, 410-2660 Oak Street, Vancouver, British Columbia, V6H 3Z6, Canada. Tel: +1 604 875 4111. Tel: x 62107. Fax: +1 604 875 4013. E-mail: [email protected] *Both authors contributed equally to this work.
(Received 7 September 2013; revised 13 October 2013; accepted 16 October 2013) ISSN 0036-5521 print/ISSN 1502-7708 online 2014 Informa Healthcare DOI: 10.3109/00365521.2013.857713
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that this pathogen is able to modulate the immune system for its own benefit. Macrophages engulf microorganisms by enclosing them in vesicles termed phagosomes, which fuse with lysosomes, organelles in charge of vesicle cargo digestion including phagosomes. MAP is able to infect, reside, and multiply in bovine and human macrophages [16–18], suggesting that the pathogen is able to elude the host’s immune response. Although the exact survival mechanism of MAP in macrophages remains to be elucidated, our previous report showed the secretion of the mycobacterial protein tyrosine phosphatase (PtpA) in a time-dependent manner on MAP infection of human-derived monocyte cell line THP-1 [19]. The function of PtpA has been elucidated in M. tuberculosis, a very similar pathogen, which also secretes PtpA and shares a 90% homology to MAP PtpA [18]. PtpA inhibited phagosome acidification [19] and the phagosome–lysosome fusion event in human macrophages by dephosphorylating the host sorting protein VPS33B [20]. By avoiding the lysosome, the pathogen escapes host immunological surveillance and establishes successful infection. We hypothesized that as in the case of M. tuberculosis, PtpA also promotes the survival of MAP in host cells, and as a secreted protein within macrophages it would be processed as foreign protein, and presented to antibody-producing cells. Then, if MAP is able to infect humans, we would expect that antibodies against PtpA are present in serum of CD patients as a result of antigen processing. Indeed, recently we found significantly higher levels of antibodies in CD patients against PtpA when compared to healthy controls [17], and as mentioned above, a decrease in the levels of antibodies against PtpA was measured in CD patients under infliximab treatment [15]. Following our previous studies, we analyzed antibody levels against PtpA in a bigger cohort of CD patients and examined the variation of these antibodies depending on the treatment provided. In addition, levels of antibodies against PtpA were measured in ulcerative colitis (UC) patients. Methods Patients The research ethics board of Mount Sinai Hospital, Toronto, Ontario, Canada, approved the protocol for this study. All subjects were recruited at Mount Sinai Hospital in Toronto, Canada between the years of 2003 and 2011 and provided written informed consent prior to sample collection. Serum samples were obtained from peripheral blood from 110 patients with a proven diagnosis of CD, 82 patients diagnosed
with UC, and 150 patients without CD or UC (healthy controls). The inclusion criteria for subjects are as follows: i) No previous history of tuberculosis infection, ii) No major risk factors for tuberculosis (HIV positive status, organ transplant recipient, known contact with TB patients, and iii) No antibiotic use which may potentially have activity against Mycobacteria in the previous 6 months. Information regarding demographic, medication history, and previous disease course were collected at study entry. Based on the clinical records obtained to perform this study, we were not able to stratify the two forms of CD (perforating and non-perforating) and the data show in this study is presented as a “single” form of CD [21]. Blood collection Peripheral blood collected from CD patients, UC patients, and controls was processed in order to obtain sera. Whole blood (5–10 ml) was collected in tubes without any additives at Mount Sinai Hospital. Each specimen was labeled with a study code and the serum was obtained after centrifugation of the coagulated whole blood at 3000 rpm for 10 min. Serum was aliquoted in cryovials with external caps and internal O-ring seals and kept at 80oC. The investigators performing the ELISA tests were blinded to the clinical characteristics associated with each specimen. Bacterial strains MAP strains K-10 (ATCC BAA-968) was cultured in Middlebrook 7H9 broth (BD) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (BD), 0.05% Tween-80 (Fisher), and 2 g/l mycobactin J (Allied Monitor). When necessary, solid medium was prepared by adding 1.5% agar (BD). MAP was cultured in rolling bottles and 0.7 108 cells were inoculated in the broth. Cultures were exposed to different concentrations of drugs currently used for CD treatments. Prednisone (Sigma-Aldrich) was dissolved in DMSO and used at final concentrations of 12.5, 62.5, 1,250 mg/ml; whereas 5-aminosalycilic acid (5-ASA) also dissolved in DMSO was used at final concentrations of 10, 25, 50, and 100 mg/ml. Bacterial growth was monitored at OD of 600 nm. Antigen Production and ELISA Recombinant PtpA was produced in M. smegmatis and according to published procedures [18]. Ninty-six-well flat bottom plates were coated with 50 mg of recombinant PtpA in 50 ml of coating buffer (0.1 M NaHCO3 pH 9.6) and placed at 4 C overnight. Next day, wells were washed (3) with phosphate buffer saline (PBS),
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Variation of MAP proteins immunogenicity in IBD patients and blocked with 200 ml of 3% bovine serum albumin (BSA) in PBS overnight at 4 C. A serum dilution of 1:100 was found to be the optimal dilution. Thus, sera were diluted 1:100 in 3% BSA, and 50 ml was added to each well. After 2 h incubation at room temperature, wells were washed (3) with PBS supplemented with 0.05% Tween-20 (PBS-T). Goat anti-human coupled to horseradish peroxidase (Jackson) was used as secondary antibody diluted 1:5000 in 3% BSA solution. After 1 h exposure, wells were washed (3) with PBS-T and 50 ml of a solution of 3,3¢,5,5¢-tetramethylbenzidine (Alfa Aesar) was used as the developer. Reactions were stopped with a solution of 25 ml of 1 M sulfuric acid. Plates were read at 450 nm using an ELISA reader. Fetal calf serum and the secondary antibody were used as a negative control. All the experiments were performed in triplicate. Statistical analysis To compare ELISA tests between CD and UC positive and healthy patients for PtpA, assuming a nonnormal distribution, Wilcoxon rank sum test was used and a Bonferroni correction for multiple comparisons was implemented. p < 0.05 was considered statistically significant. Results Antibodies against PtpA in sera of CD and UC patients A total of 342 archived samples corresponding to 110 CD, 82 UC patients, and 150 controls were
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analyzed in this study. All CD and UC patients had well-characterized disease based on clinical, endoscopic, radiologic, and histological criteria (Table I). When sera of CD and UC patients and controls were screened for antibodies against PtpA, a mean antibody intensity reading of 0.232 and 0.145 were calculated for CD and UC patients, respectively, whereas a mean of 0.108 was measured in the control group (Figures 1 and 2). CD (p < 0.0001) but not UC patient(p = 0.3124) sera showed significantly higher titer of antibodies against PtpA when compared to controls.
Effect of CD and UC treatments on the level of antibodies against PtpA The impact of usual therapies typically taken by patients with IBD include azathioprine (AZA), 5-ASA, infliximab, and steroids including both prednisone and budesonide. We were interested in determining whether treatments with these medications alter levels of antibodies against PtpA, because our previous studies found that CD patients treated with infliximab had significantly decreased levels of PtpA antibodies. Variability in the level of antibodies against PtpA was observed when sera from treated patients were analyzed. A statistical difference was observed between CD patients treated with AZA and untreated CD patients, whereas no significant difference was observed when patients were treated with either 5-ASA or steroids (Figure 2). Interestingly, when AZA was combined with other medications, such as 5-ASA and/or steroids, no significant difference was measured (Figure 2).
Table I. Clinical characteristics of study patients (n = 342).
Age in year, median (range) Gender, female n (%) Years of disease, median ± SD Medical therapy, n (%) Prednisone Azathioprine Methotrexate Budesonide 5-ASA Combined* None Previous surgeries, n (%) Perianal fistula Colostomy Hemi colostomy Ileal resection Ileocolonic resection Small bowel (other than ileum) resection
CD patients (n = 110)
UC patients (n = 82)
Healthy controls (n = 150)
32 (17–81) 51 (46) 8.5 ± 8.8
34 (23–53) 38 (46) 8 ± 7.8
38 (17–85) 79 (53) n/a
8 (10) n/a n/a 1 (1) 34 (41) 21 (26) 18 (22)
n/a n/a n/a n/a n/a n/a n/a
n/a n/a n/a n/a n/a n/a
n/a n/a n/a n/a n/a n/a
6 16 3 9 8 31 37
(5) (14) (3) (8) (7) (28) (33)
1 (0.9) 1 (0.9) 4 (4) 28 (25) 1 (0.9) 3 (3)
Abbreviation: n/a = non applicable. *Combined, a combination with at least two of the listed drugs.
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Figure 1. Distribution of the antibody intensity readings. A boxplot analysis shows the distribution of the antibody intensity readings obtained by ELISA in untreated CD and UC patients. *p-Values of 0.0009 and < 0.001 were obtained using Wilcoxon rank sum test and Bonferroni test, respectively. OD = optical density; HC = healthy controls; CD = Crohn’s disease; UC = Ulcerative colitis. Experiments were performed in triplicate.
In the case of UC patients, no differences in PtpA antibody levels were observed when patients were treated with AZA, 5-ASA, and steroids, singly or combined (Figure 3). Exposure of MAP to 5-ASA and prednisone Previous studies reported impaired growth of MAP when it was cultured in the presence of AZA [22] or infliximab [15], suggesting that exposure of MAP to these drugs affects the normal physiology of the pathogen in vitro. To determine the effect of 5-ASA and steroids on MAP growth, the pathogen was exposed to different concentrations of both compounds in vitro. In both cases, an increase in MAP proliferation was recorded, indicating that these compounds have no detrimental effect in the normal growth of MAP (Figure 4A, B). In the case of 5-ASA, MAP growth was intensified with exposed to a concentration of >25 mg/ml when compared to the non-treated control (Figure 4A).
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Figure 2. Distribution of the antibody intensity readings in CD untreated and treated patients. A boxplot analysis shows the distribution of the antibody intensity readings obtained by ELISA in untreated CD patients, CD patients under individual treatment with azathioprine (AZA) (A), 5-ASA (M), and steroids (S) or their combinations (A+M+S). *p-Values of 0.0032 < 0.001 were obtained using Wilcoxon rank sum test and Bonferroni test, respectively. OD = optical density; CD = Crohn’s disease. Experiments were performed in triplicate.
CD patients in comparison to controls in small cohorts [15,17]. Moreover, levels of antibodies in CD patients treated with infliximab showed no statistical differences when compared to controls,
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Discussion To explore whether current treatments for CD cause variability in the level of antibodies against PtpA, pilot analysis was conducted on archived serum samples to evaluate the presence of these antibodies. Sera of untreated UC patients, as well as UC patients under different treatments, were also tested. We have shown in two previous studies that levels of antibodies against PtpA were significantly higher in
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Figure 3. Distribution of the antibody intensity readings in UC untreated and treated patients. A boxplot analysis shows the distribution of the antibody intensity readings obtained by ELISA in untreated UC patients, UC patients under individual treatment with 5-ASA (M), and steroids (S) or their combinations (A+M+S). OD = optical density; UC = ulcerative colitis. Experiments were performed in triplicate.
Variation of MAP proteins immunogenicity in IBD patients B
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Figure 4. Growth curve of MAP exposed to (A) 5-ASA and (B) prednisone. MAP cultures were exposed to increasing concentrations of 5-ASA and prednisone and sampled at specific time points. The optical density of the culture was measured at 600 nm. Shown are the mean values (±SE) of three independent experiments.
suggesting that inhibition of TNF-a was detrimental either to the survival of the pathogen or to the establishment of a successful infection, or that infliximab treatment affects the antigen presentation system of macrophages. In this study, a significant difference in the levels of antibodies against PtpA in CD-treated versus nontreated patients was observed (Figure 1). These results are in concord with our preliminary study performed in a smaller cohort [17] and reinforce the role this novel antibody can play in assessing patient’s exposure to MAP. Interestingly, variability in the levels of the antibodies against PtpA was observed when CD patients were subjected to different treatments. For instance, in this study, sera from CD patients treated with AZA had decreased levels of antibodies; while sera from 5-ASA or prednisonetreated CD patients did not show any significant difference in antibody levels. Studies on the effects of different non-antibiotic drugs, such as 5-ASA [23], AZA [22], cyclosporine A [24], methotrexate [25], rapamycin [24], tacrolimus [24], and thalidomide [26] on MAP growth have been reported. The immunosuppressant AZA appears to inhibit MAP growth in vitro [22] and in our study, a decrease in the level of antibodies against PtpA was measured in CD patients treated with this drug. This finding suggests that if AZA inhibits the proliferation of MAP, a concomitant decrease in the levels of antibodies against PtpA would be observed due to the reduction of MAP cells. On the other hand, patients treated with 5-ASA or steroids showed no significant differences in antibody levels when compared to untreated CD patients, suggesting that if MAP is active in CD patients, these treatments have no effect on the pathogen. Interestingly, although the use of AZA decreases the levels of antibodies against PtpA, when it is combined with other drugs, these effects are attenuated as each individual drug in the combination may
have a different impact in the regulation of signal transduction pathways. For example, it has been reported that 5-ASA possesses scavenging and antioxidant effects against reactive oxygen and nitrogen species [27]. In addition, 5-ASA activates the production of heme-oxygenase-1, which converts heme into biliverdin releasing carbon monoxide (CO). This particular CO has anti-inflammatory properties including down regulation of the expression of inflammatory cytokines [28,29]. Taking together, the presence of 5-ASA in the drug combination would provide a benefit to MAP because of its anti-inflammatory, antioxidant, and scavenging properties. In the case of 5-ASA, a previous study showed that inhibition of MAP growth was observed when bacilli were exposed to 5-ASA in vitro [23]. The results presented here related to the level of antibodies provide contradictory results because no effects on the antibody levels were observed in patient sera treated with 5-ASA. Differences in the results can be explained by either a difference in MAP species being used or the special cell morphology (spheroplast-like cells) of MAP when it is isolated from human tissues. Since spheroplast MAP cells lack a cell wall, a remodeling of the bacterial surface is expected and drugs can be beneficial or detrimental for MAP growth. Therefore, inhibition of MAP by 5-ASA in vivo would be different when compared to in vitro conditions when the whole cell wall is present [30]. No previous studies have examined the effect of steroids in MAP. In this report, we show that the steroid prednisone does not affect MAP growth. A previous report describing the proliferation of MAP in hamsters treated with the steroid dexamethasone showed significantly higher bacterial counts in spleen and Peyer’s patches [31]. In addition, Mycobacteria are able to degrade cholesterol [32] and the pathogenic M. tuberculosis is able to use cholesterol as a unique carbon source [33]. Since, steroids used for treatments are derivatives of cholesterol and taking in account that
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MAP possesses all the enzymes to degrade cholesterol, steroids can be exploited by the pathogen as a carbon source and that can explain why a higher number of MAP colonies was observed in hamsters treated with the steroid dexamethasone. Results reported here show that significant levels of antibodies against PtpA were not measured in untreated UC patients, suggesting that MAP is not involved in UC etiology. This conclusion, although controversial, is supported by several reports that showed differential findings of MAP in CD and UC patients. For example, a meta-analysis study performed to assess the association of MAP with CD found an association with MAP in CD, but not in UC [34]. Another study reported the amplification of the DNA fragment IS900, commonly used for the specific detection of MAP, in 52% tissue samples from CD patients and only 2% in specimens obtained from UC patients [4]. On the other hand, some studies have reported inconclusive results as no differences in antibodies against MAP antigens were observed among the study groups including CD and UC patients, healthy controls, and non-affected siblings [35]. However, 15% of 29 UC patients reacted positively with a MAP antigen [36] and MAP DNA was detected in 20% of UC patients, but in 33% of healthy controls, suggesting that MAP is widely distributed in the environment including in the food chain [37]. Interestingly, a study performed in Manitoba, Canada found no differences in the association between MAP and healthy controls, CD or UC patients [38]. Results of this study were performed by adapting an ELISA test used by veterinarians to diagnose JD. One explanation for this difference is the changes that MAP undergoes when infecting the human host. Physiological changes render this pathogen devoid of the cell wall, probably as a way to escape from the immune system [3]. Thus, fewer antigens will be available to prime the immune system and fewer antibodies will be measured. A second possible explanation is the fact that the province of Manitoba possesses high incidence and prevalence rates of CD [39]. Thus, the presence of antibodies can be related to an exposure to the pathogen rather than an actual infection. In our case, we measured the antibodies against a virulence factor of the pathogen, which is secreted in a constant manner only within the host. Our results also concord with other studies where differences between CD patients and healthy controls were found using the MAP antigens p35 and p36 [36]. In conclusion, we found different levels of PtpA antibodies against MAP in human sera, which vary according to the medical treatment of CD patients, but no significant differences were observed in UC
patients, suggesting that MAP is not associated with this disease. Serial measurements of PtpA antibodies in patients with CD treated with various medications may be another outcome that should be followed to optimize medical care. Further research will be done to understand this novel approach to CD treatment.
Acknowledgement We are very thankful to Jeffrey Helm for helpful discussion. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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