Abstract
Background
Predicting the response of inflammatory bowel disease (IBD) patients to infliximab (IFX) is an unmet clinical need. The expression and density of transmembrane tumor necrosis factor-α in circulating leukocytes maybe directly related to response by promoting apoptosis.
Aim
We tested the hypothesis that direct apoptosis assessment by real-time polymerase chain reaction evaluation of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins in peripheral blood mononuclear cells (PBMCs) might be associated with response to IFX.
Methods
IFX naïve patients (Crohn’s disease, 32 and ulcerative colitis, 20; 35 responders and 17 non-responders) were evaluated for Bax and Bcl-2 mRNA expression levels before and 2 weeks after the first infusion. In a subset of patients, apoptosis was also evaluated using flow cytometry.
Results
After the first infusion, Bax increased more in responders than in non-responders (0.7± 0.38 vs 0.81 ± 0.32 and 0.86 ± 0.37 vs 0.87 ± 0.45, respectively, p = 0.071). Bcl-2 decreased more in responders than in non-responders (0.71 ± 0.12 vs 0.63 ± 0.13 and 0.81 ± 0.28 vs 0.77 ± 0.27, respectively, p = 0.038). The Bax/Bcl-2 ratio increased more in responders than in non-responders (0.99 ± 0.5 vs 1.3 ± 0.51 and 1.03 ± 0.17 vs 1.1 ± 0.28, respectively, p = 0.005). The Bax/Bcl-2 ratio was able to predict response in 33/52 patients and was correlated to flow cytometry-assessed apoptosis (r = 0.911; p < 0.001).
Conclusions
An increased Bax/Bcl-2 ratio in PBMCs was associated with therapeutic response to IFX in IBD patients.
Keywords: Apoptosis, inflammatory bowel diseases, infliximab, therapeutic response
Key summary
What is known:
The identification of biomarkers to predict the response of inflammatory bowel disease patients to infliximab is an unmet clinical need.
We have shown that the expression and density of transmembrane tumor necrosis factor-α in circulating leukocytes maybe directly related to the primary response by promoting apoptosis.
Measurement of apoptosis by traditional means (flow cytometry) is expensive and often not available in clinical laboratories.
Main findings of the study:
We show that the Bax/Bcl-2 ratio (the ratio of pro-/anti-apoptotic proteins), as measured by an easily available technique (real-time polymerase chain reaction), in peripheral blood cells is able to predict response to infliximab in the majority of inflammatory bowel disease patients and is correlated to the apoptosis rate assessed by flow cytometry.
This method could potentially be used as a clinical biomarker for primary response to infliximab.
Introduction
Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic gastrointestinal disorders that are possibly caused by a complex interaction of environmental, genetic and immunoregulatory factors.1 For both CD and UC, the biological anti-tumor necrosis factor-α (TNF-α) agents infliximab (IFX), adalimumab and certolizumab have been proven very effective in inducing and maintaining clinical remission over the last 18 years. However, these medications can cause side effects and are expensive.2,3 More importantly, a subgroup of IBD patients do not respond to anti-TNF agents (primary non-responders (PNRs)) or loose response over time (secondary non-responders).4 While lack/loss of response might be due to a number of reasons,5 early identification of non-responders might avoid their exposure to potential side effects and reduce the costs of therapy.6 In addition, the identification of the mechanism(s) behind the lack/loss of response might offer crucial clues for the development of new medications.
Transmembrane TNF-α (tmTNF-α), a precursor of the soluble form of TNF-α (sTNF-α), is expressed on the surface of a number of activated inflammatory cell types. Interaction of IFX and tmTNF-α triggers several anti-inflammatory responses including tmTNF-α reverse signaling-induced apoptosis, which subsequently results in the depletion of inflammatory cells and the attenuation of inflammation, a mechanism that appears to be the basis of the well-known clinical efficacy of these agents.6,7
A recent study from our group showed that the percentage of tmTNF-α-bearing lymphocytes and monocytes and the tmTNF-α density in circulating leukocytes are directly related to the primary response to IFX through the promotion of inflammatory cell death in IBD patients. Hence, peripheral leukocytes could, in principle, be used to predict primary response to IFX in IBD patients.8 However, evaluation of tmTNF-α density requires methods such as flow cytometry, which are not easily applicable in routine clinical practice.
The mitochondrial-mediated pathway of apoptosis is regulated by the Bcl-2 family of anti-apoptotic (Bcl-2, Bcl-xl and Mcl-1) and pro-apoptotic proteins (Bax, Bad and Bak).9 It has been shown that the Bax/Bcl-2 ratio may be more important than either protein alone in determining apoptosis,10 since an increased Bax/Bcl-2 ratio upregulates caspase-3, which in turn increases apoptosis.11 Bax, Bcl-2 and the Bax/Bcl-2 ratio can be evaluated using real-time polymerase chain reaction (RT-PCR), a common laboratory method. Hence, in this study, we tested the hypothesis that direct evaluation of apoptosis by RT-PCR in circulating inflammatory cells could potentially be used as an early marker of response to IFX, following the first drug infusion.
Materials and methods
The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in the approval by the Institutional Review Board of Shahid Beheshti University of Medical Sciences (date of approval 21 December 2015, under the ethical code of IR.SBMU.RIGLD.REC.135.48/49). Written consent was obtained from each patient before enrolment in the study.
The patient population, consisting of IFX naïve IBD patients who failed corticosteroids and/or immunomodulators, as well as the inclusion and exclusion criteria have been previously described.8 A total of 63 patients were identified as eligible to be included in the study. Six and five patients dropped out because of consent withdrawal and intolerance to IFX, respectively. Hence, a total of 52 patients entered and completed the study (Figure 1).
Figure 1.
Flowchart of the study design with inclusion/exclusion criteria for inflammatory bowel disease patients.
FC: fecal calprotectin; CRP: C-Reactive protein; RT-PCR: real-time polymerase chain reaction; IFX: infliximab.
The treatment consisted of standard IFX dose (5 mg/kg) at weeks 0, 2 and 6, and every 8 weeks thereafter. A 10 ml blood sample was taken from each patient at baseline and at week 2 before the second IFX infusion. Peripheral blood mononuclear cells (PBMCs) were extracted by gradient centrifugation on a layer of Ficoll-Paque Plus® (Amersham, Uppsala, Sweden) and stored at −80℃ for subsequent tests.
Evaluation of Bax, Bcl-2 and Bax/Bcl-2 ratio using relative quantitative RT-PCR
Following the RNA extraction of samples using a YTA RNA Extraction kit (YektaTajhiz Azma, Tehran, Iran) and treatment of the extracted RNA with DNase, complementary DNA (cDNA) was synthesized using a Revert Aid First Strand cDNA Synthesis kit according to the manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA). β-2microglobulin (B2M) was used as the reference gene. Then, the primers were designed and synthesized. Their sequences were as follows: Bax F, 5′-CCTGTGCACCAAGGTGCCGGAACT-3′; Bax R, 5′-CCACCCTGGTCTTGGATCCAGCCC; Bcl-2 F, 5′-TTGTGGCCTTCTTTGAGTTCGGTG-3′; Bcl-2 R: 5′-GGTGCCGGTTCAGGTACTCAGTCA-3′; B2M F: 5′-TGCTGTCTCCATGTTTGATGTATCT-3′ and B2M R: 5′-TCTCTGCTCCCCACCTCTAAGT-3′.
Subsequently, RT-PCR was performed using SYBR® Premix Ex Taq™ II (Takara, Japan) on a StepOnePlus™ RT-PCR System (Applied Biosystems, Waltham, MA, USA) for 1 cycle at 95℃ for 2 min, followed by 40 cycles at 95℃ for 5 s and 60℃ for 30 s. Specificity of the products was verified by melting curve analysis (Fig.1S). Each assay was done in triplicate. Data were analyzed using the comparative Ct method. In this respect, 2−ΔCt (where ΔCt is CT gene of interest − CT internal control) was calculated for each sample and used for further statistical analysis.12 Since the basic assumption of the comparative Ct method is the approximately equal amplification efficiency of target and reference genes, the efficiency for each gene was calculated using real-time LinRegPCR software for all samples in each PCR run and for those with inconsistent efficiency when the test was repeated.
Apoptosis evaluation using flow cytometry
For comparison purposes, the apoptosis rate in PBMCs was also evaluated in a subset of patients (16 responders and 10 non-responders) using flow cytometry, as previously described.8 Freshly isolated mononuclear cells were used as recommended.13 The cells were first stained by a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (BioLegend, San Diego, CA, USA) with propidium iodide. Then, the cells were washed twice with cold BioLegend's cell staining buffer and resuspended in annexin V binding buffer. After the addition of the FITC, annexin V and propidium iodide solution, and 15 min incubation at room temperature in the dark, annexin V binding buffer was added. The cells then underwent flow cytometry. Negative and positive controls were also included. Immediately after this step, flow cytometric analysis was performed on a FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). At least 10,000 PBMCs per sample were evaluated. The data were analyzed using Flow Jo software (TreestarInc, Ashland, OR, USA) (Fig.2S).
Evaluation of the response to IFX
The common clinical definition of response to IFX is the improvement of clinical signs and symptoms after induction therapy.14 However, we chose to evaluate the response more objectively, using endoscopic disease activity (Crohn’s disease endoscopic index of severity (CDEIS) for CD and Mayo score for UC)15,16 before and after IFX infusion. C-reactive protein (CRP), and fecal calprotectin (FC) (FCal enzyme-linked immunosorbent assay, Bühlmann, Schönenbuch, Switzerland) were also evaluated at weeks 0 and 14.
Statistical analysis
IBM SPSS for Windows, version 16 was used for all statistical analysis. The Student’s t-test or the Mann–Whitney U test was used for the evaluation of the mean difference of variables before and after IFX therapy. The analysis of covariance test was used to analyze the mean difference between the two groups with treatment as factor and baseline as covariates. Pearson’s or Spearman’s correlation coefficient was used to evaluate potential correlation between variables. The prognostic value of candidate variables was evaluated by plotting receiver operating characteristic (ROC) curves and calculation of the area under the curve (AUC). The maximum point of Youden's index (Youden's J) was identified as the cutoff value in the separation of responders and non-responders. Median split was used to dichotomize quantitative variables such as age and disease duration. The χ2 test was used to evaluate individual patient/disease features in the prediction of response by the Bax/Bcl-2 ratio. A p-value of ≤0.05 was considered statistically significant.
Results
A total of 52 IFX naïve IBD patients were included in the study. The patient population was composed of 32 CD and 20 UC patients, with 26 females and 26 males (mean age 35.9 ± 13.2 years, range 17–60; mean disease duration 6.5 ± 2 years, range 3–12). A positive smoking history was observed in 5 (9.6%) of the patients. The clinical and demographic characteristics of the patients classified by disease type are shown in Table 1.
Table 1.
Baseline characteristics of inflammatory bowel disease patients.
| Variable | Results |
|
|---|---|---|
| CD (n = 32) | UC (n = 20) | |
| Mean age (mean ± SD) years | 34.7 ± 13.4 | 37.7 ± 13 |
| Gender (%) | ||
| • Male | 17 (53.1) | 9 (45) |
| • Female | 15 (46.9) | 11 (55) |
| Smoking history (%) | ||
| • Positive | 2 (6.3) | 3 (15) |
| • Negative | 30 (93.7) | 17 (85) |
| Family history of IBD (%) | ||
| • Positive | 6 (18.8) | 2 (10) |
| • Negative | 26 (81.2) | 18 (90) |
| Mean disease duration (mean ± SD) years | ||
| Classification: | 6.6 ± 2.1 | 6.3 ± 1.8 |
| L1, ileal (%) | 10 (31) | – |
| L2, colonic (%) | 9 (28) | – |
| L3, ileocolonic (%) | 13 (41) | – |
| Classification: | ||
| E1, rectum (%) | – | – |
| E2, left-sided (%) | – | 9 (45) |
| E3, extensive (%) | – | 11 (55) |
CD: Crohn’s disease; UC: ulcerative colitis; IBD: inflammatory bowel disease.
Based on response to IFX (according to the criteria described in the Materials and Methods), we identified a total of 35 (67.3%) responders and 17 (32.7%) non-responders (Table 2). The mean baseline endoscopic scores (CDEIS and Mayo) were similar in responders and non-responders. At week 14 (after induction), the mean CDEIS score decreased by 8.4 points in responders and by 1.6 points in non-responders (p < 0.001). The mean Mayo subscore decreased by 1.3 points in responders and increased by 0.2 points in non-responders (p < 0.001).
Table 2.
Comparison of clinical and laboratory characteristics of infliximab responders and non-responders.
| Variable | Responders |
Non-responders |
p-value | ||
|---|---|---|---|---|---|
| CD (%) | UC (%) | CD (%) | UC (%) | ||
| Disease type | 21 (60) | 14 (40) | 11 (64.7) | 6 (35.3) | – |
| 35 (67.3) | 17 (32.7) | ||||
| CDEIS week 0 | 17.7 ± 4.8 | – | 18.3 ± 4.0 | – | <0.001 |
| CDEIS week 14 | 9.3 ± 3.5 | – | 16.7 ± 4.3 | – | |
| Mayo endoscopic subscore week 0 | – | 2.6 ± 0.5 | - | 2.3 ± 0.5 | < 0.001 |
| Mayo endoscopic subscore week 14 | – | 1.3 ± 0.5 | - | 2.5 ± 0.6 | |
| CRP (mg/L) week 0 | 41.6 ± 40 | 37.2 ± 36 | < 0.001 | ||
| CRP (mg/L) week 14 | 5.8 ± 4.4 | 30.6 ± 33 | |||
| FC (µg/g) week 0 | 601 ± 404 | 581 ± 389 | <0.001 | ||
| FC (µg/g) week 14 | 134 ± 100 | 648.7 ± 412 | |||
Data are shown as N (%) or mean ± SD.
CD: Crohn’s disease; UC: ulcerative colitis; CDEIS: Crohn's disease endoscopic index of severity; CRP: C-reactive protein; FC: fecal calprotectin.
Mean baseline CRP and FC were similar in responders and non-responders. However, at week 14, mean CRP decreased by 35.8 mg/L in responders and by 6.6 mg/L in non-responders (p < 0.001), and mean FC decreased by 467 µg/g in responders and increased by 67.7 µg/g in non-responders (p < 0.001).
Bax and Bcl-2 expression, as well as the Bax/Bcl-2 ratio, were measured in both responders and non-responders in samples taken at baseline (week 0) and at week 2 (immediately before the second IFX infusion).
Mean baseline Bax expression level was 0.7 ± 0.38 in responders and 0.86 ± 0.37 in non-responders. Two weeks after the first IFX infusion, it increased to 0.81 ± 0.32 in responders and to 0.87 ± 0.45 in non-responders (p = 0.071) (Figure 2(a)).
Figure 2.
(a) Comparison of mean Bax mRNA levels between the peripheral blood mononuclear cells of infliximab responders and non-responders. (b) Comparison of mean Bcl-2 mRNA levels between the peripheral blood mononuclear cells of infliximab responders and non-responders. (c) Comparison of mean Bax/Bcl-2 mRNA ratio between the peripheral blood mononuclear cells of infliximab responders and non-responders; Data are shown as mean ± SD. (*) shows a significant p-value (≤0.05).
Mean baseline Bcl-2 expression level was 0.71 ± 0.12 in responders and 0.81 ± 0.28 in non-responders. Two weeks after the first IFX infusion it decreased to 0.63 ± 0.13 in responders and to 0.77 ± 0.27 in non-responders (p = 0.038) (Figure 2(b)).
The Bax/Bcl-2 ratio increased from 0.99 ± 0.5 at baseline to 1.3 ± 0.51 at 2 weeks in responders and from 1.03 ± 0.17 to 1.1 ± 0.28 in non-responders, respectively (p = 0.005) (Figure 2(c)).
The mean apoptosis percentage as measured by flow cytometry at week 2 was 3.3 in responders and 2.2 in non-responders (p = 0.06). A significant positive correlation was observed between the Bax/Bcl-2 ratio at week 2 and the apoptosis rate (r = 0.911; p < 0.001) (Figure 3).
Figure 3.
Scatter plot (Spearman’s correlation) showing a strong positive correlation between the Bax/Bcl-2 mRNA ratio and the apoptosis percentage in the peripheral blood mononuclear cells of inflammatory bowel disease patients, 2 weeks after the first infliximab infusion.
ROC curves and calculation of the AUC were used to evaluate the predictive power of the Bax/Bcl-2 ratio at week 2 in separating responders from non-responders. An AUC of 0.636 (95% confidence interval (CI), 0.479–0.0787; p = 0.12) with an optimal cutoff value of 1.2, and a sensitivity and specificity of 57% and 76.5%, respectively, was obtained (Figure 4).
Figure 4.
Receiver operating characteristic curve showing the predictive power of the Bax/Bcl-2 mRNA ratio of peripheral blood mononuclear cells in distinguishing infliximab responders and non-responders 2 weeks after the first infliximab infusion.
ROC: receiver operating characteristic; AUC: area under the curve; CI: confidence interval.
Based on the cutoff value, the Bax/Bcl-2 ratio was able to correctly predict response in 2/3 (33/52) of IBD patients (20/35 responders and 13/17 non-responders). When adjusted according to the disease type, the Bax/Bcl-2 ratio correctly predicted response in 68.7% of CD patients and in 55% of UC patients (p = 0.2). No significant association was found between correct prediction of response by the Bax/Bcl-2 ratio and patient/disease features such as gender, age (<or >37 years), smoking history (yes/no), disease duration (< or >6.5 years), baseline endoscopic scores (CDEIS < or > 18.5; Mayo score < or > 3) and baseline inflammatory markers (CRP < or > 19.5 mg/L; FC: < or > 660 µg/g).
Discussion
For more than a decade, IFX has been used for the treatment of refractory IBD with up to 70% of IBD patients showing significant clinical improvement with this therapy.17 Due to the cost and potential side effects of IFX, the development of a biomarker capable of predicting response to IFX prior to its infusion would be of considerable value.
To date, several potential biomarkers, such as pharmacogenetic biomarkers,18 sTNF-α levels,19 and others,20 have been tested. We have recently shown that the percentage of tmTNF-α-bearing lymphocytes and monocytes and the intensity of tmTNF-α in the circulating leukocyte population of IBD patients is directly related to primary response to IFX.8 This is likely due to the promotion of inflammatory cell death (apoptosis). Indeed, defects in programmed cell death of the mucosal T cell compartment have been suggested to play a role in the pathogenesis of IBD,21,22 and the efficacy of anti‐TNF agents has been shown to be at least partially due to its ability to induce apoptosis in defective T cells and monocytes.23–25
While the susceptibility of PBMCs to IFX has been less well studied, evidence suggests that IFX treatment could induce apoptosis in the PBMC population as well.26–29
Indeed, in our previous study, we showed that the mean percentage of apoptosis in PBMCs was significantly greater in responders compared to non-responders.8 Expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins, the main members of the apoptotic pathway, have been widely used in the evaluation of apoptosis and it has been widely reported that the Bax/Bcl-2 ratio is a reliable marker of the apoptosis rate in several conditions.11,30–32 Indeed, Eder et al. have observed a significant increase in pro-apoptotic Bax/Bcl-2 ratio in lamina propria mononuclear cells of CD patients treated with anti-TNF agents and have concluded that the efficacy of these medications is at least partly dependent on apoptosis modulation.33
Here, we have tested the hypothesis that assessment of the Bax/Bcl-2 ratio by RT-PCR, a common laboratory method,34 in circulating inflammatory cells could potentially be used as an early, inexpensive and clinically useful marker of primary response to IFX, following the first drug infusion.
The results show that 2 weeks after the first IFX infusion, the Bax/Bcl-2 ratio in the PBMCs of responders was significantly greater compared to non-responders. By plotting ROC curves and calculation of the AUC, we established that a Bax/Bcl-2 ratio cutoff of 1.2 was able to correctly predict response in two-thirds (33/52) of IBD patients. A significant positive correlation was observed between the Bax/Bcl-2 ratio at week 2 and the apoptosis rate as measured by flow cytometry, which further validates the data obtained by RT-PCR.
Hence, our data indicate that the Bax/Bcl-2 ratio in PBMCs could be potentially used as a convenient biomarker for prediction of response to IFX in the majority of IBD patients. The fact that the Bax/Bcl-2 ratio fails to correctly identify all the IFX responders/non-responders might be due to a number of reasons, including the multifactorial pathogenesis of IBD35 as well as the complex, multitasker anti-inflammatory role of IFX.36 Thus, it is possible that pathways (untested in this study) that do not involve apoptosis might play a role in determining the therapeutic response to IFX in a proportion of IBD patients.
Van den Brande et al. also measured apoptosis to predict response to IFX in patients with Crohn's disease. These authors used the annexin V scan method for real‐time visualization of apoptosis. They found that colonic uptake of 99mTc-annexin V is associated with clinical efficacy of IFX and might be predictive of therapeutic success.37 Hence, our data are consistent with those of Van den Brande et al.37 In comparison with the annexin V scan method, RT-PCR is simpler (it only needs a single blood test), cheaper and does not require radioactive substances. Furthermore, it requires little technical skill and no dedicated facilities.
The Bax/Bcl-2 ratio might also be potentially useful in monitoring the safety and efficacy of IFX in subtle decision-making scenarios, such as its initial administration during pregnancy,38 which adds complexity to the management of IBD.
Although the majority of studies have demonstrated an increased apoptosis rate in peripheral blood cells of IBD patients following IFX treatment,26,37 some studies, such as the study of Hove et al., reached different conclusions.39 This inconsistency could be attributed to the characteristics of the individual studies, which targeted different cell types.
Clearly, the relatively small sample size of our study limits the generalizability of our findings. However, it should be pointed out that, by contrast with most other studies, IFX response was defined by strict and objective criteria and our patient population truly represented IFX PNRs.
In conclusion, an increased Bax/Bcl-2 ratio in PBMCs of IBD patients was associated with therapeutic response to IFX. This method could potentially be used as a clinical biomarker of primary response. Since the Bax/Bcl-2 ratio was unable to predict IFX response in all patients, it is possible that pathways other than apoptosis might mediate the anti-inflammatory action of IFX in IBD.
Declaration of conflicting interests
D.S. has received consulting fees from Abbott/AbbVie, Schering-Plough, MSD, Janssen Research & Development, LLC, Centocor Inc., TechLab, Hoffmann-LaRoche, Giuliani, Schering-Plough and Ferring; research grants from AbbVie, Janssen Research & Development, LLC, Schering-Plough, TechLab and Centocor; and serves in the Speakers Bureau of AbbVie and the National Faculty of Janssen and Medtronic. The other authors have no conflicts of interest to declare.
Funding
This study has been supported by the Research Institute for Gastroenterology and Liver Diseases at the Shahid Beheshti University of Medical Sciences.
Ethics approval
The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in the approval by the Institutional Review Board of Shahid Beheshti University of Medical Sciences (date of approval 21 December 2015, under the ethical code of IR.SBMU.RIGLD.REC.135.48/49).
Informed consent
Written consent was obtained from each patient before enrollment in the study.
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