Serum Immune Profiling in Paediatric Crohn's Disease Demonstrates Stronger Immune Modulation With First-Line Infliximab Than Conventional Therapy and Pre-Treatment Profiles Predict Clinical Response to Both Treatments

Abstract

Background: Despite its efficacy, rational guidance for starting/stopping first-line biologic treatment in individual paediatric Crohn's disease [CD] patients is needed. We assessed how serum immune profiles before and after first-line infliximab [FL-IFX] or conventional [CONV] induction therapy associate with disease remission at week 52.

Methods: Pre- [n = 86], and 10-14-week post-treatment [n = 84] sera were collected from patients with moderate-to-severe paediatric CD in the TISKids trial, randomized to FL-IFX [n = 48; five 5-mg/kg infusions over 22 weeks] or CONV [n = 43; exclusive enteral nutrition or oral prednisolone]; both groups received azathioprine maintenance. The relative concentrations of 92 inflammatory proteins were determined with Olink Proteomics; fold changes [FC] with |log2FC| > 0.5 after false discovery rate adjustment were considered significant.

Results: FL-IFX modulated a larger number of inflammatory proteins and induced stronger suppression than CONV; 18/30 proteins modulated by FL-IFX were not regulated by CONV. Hierarchical clustering based on IFX-modulated proteins at baseline revealed two clusters of patients: CD-hi patients had significantly higher concentrations of 23/30 IFX-modulated proteins [including oncostatin-M, TNFSF14, HGF and TGF-α], and higher clinical disease activity, C-reactive protein and blood neutrophils at baseline than CD-lo patients. Only 24% of CD-hi FL-IFX-treated patients maintained remission without escalation at week 52 vs 58% of CD-lo FL-IFX-treated patients. Similarly, 6% of CD-hi CONV-treated patients achieved remission vs 20% of CONV-treated CD-lo patients. Clustering based on immune profiles post-induction therapy did not relate to remission at week 52.

Conclusion: FL-IFX leads to stronger reductions and modulates more immune proteins than CONV. Stratification on pre-treatment profiles of IFX-modulated proteins directly relates to maintenance of remission without treatment escalation.

Trial registration number: NCT02517684.

Keywords: Proteomics; anti-TNF; inflammatory bowel disease.

Graphical Abstract

Figure 1.

First-line infliximab [FL-IFX] induces greater qualitative and quantitative differences in serum immune protein concentrations than conventional [CONV] induction treatment. [A] Summary of trial protocol. Untreated patients received either five infusions of 5 mg/kg infliximab at weeks 0, 2, 6, 14 and 22 [FL-IFX], or CONV treatment consisting of exclusive enteral nutrition [EEN] or oral prednisolone [1 mg/kg, maximum 40 mg]. Both groups received azathioprine maintenance therapy. Serum was collected at baseline [week 0] and weeks 10–14. The endpoint was clinical remission [wPCDAI < 12.5 at week 52, without treatment escalation]. [B–D] Volcano plots of the changes in the concentrations of 83 proteins after 10–14 weeks of induction treatment. Wilcoxon signed-rank test and Mann–Whitney U-test; corrected for false discovery rate [FDR] based on the Benjamini–Hochberg procedure; p  < 0.05 was considered significant. [B] FL-IFX significantly reduced 28 proteins and enhanced two proteins [paired samples; n = 46; Wilcoxon signed-rank test]. [C] CONV significantly reduced 13 proteins and enhanced one protein [paired samples; n = 32; Wilcoxon signed-rank test]. [D] FL-IFX [n = 46] led to significantly greater reductions in the concentrations of 18 proteins compared to CONV [n = 32; Mann–Whitney U-test]. [E] Venn diagram of the 33/83 proteins significantly regulated by FL-IFX or CONV, and the six related pathways [for protein descriptions also see Supplementary Table S3]; 19 proteins were only modulated by FL-IFX; three proteins were only modulated by CONV; and 11 proteins were modulated by both treatments, but more strongly reduced by FL-IFX than CONV. A total of 30 proteins were significantly modulated by FL-IFX.

Figure 2.

First-line infliximab [FL-IFX] reduces proteins in the IFN-γ, neutrophil and IL-17, cell cycle and apoptosis, and tissue remodelling pathways. [A] Fold changes [FC] in the median serum concentrations of 83 immune proteins between baseline and after 10–14 weeks of FL-IFX or conventional [CONV] induction therapy, and differential FCs after FL-IFX and CONV at 10–14 weeks. FL-IFX induced greater qualitative [i.e. different proteins] and quantitative [i.e. the same proteins, but stronger reductions] changes than CONV. Wilcoxon signed-rank test and Mann-Whitney U-test with false discovery rate [FDR] correction based on the Benjamini–Hochberg procedure. [B–E] Comparison of the changes in protein concentrations between baseline and 10–14 weeks for the individual patients in both groups. Mann–Whitney U-test. [B] Proteins represented in the IFN-γ pathway were reduced more significantly by FL-IFX than CONV; both interferon-gamma [IFN-γ] and interleukin [IL]-18 were reduced more significantly by FL-IFX [p = 0.036 and 0.003, respectively]. [B] Proteins in the neutrophil and IL-17 pathway were significantly regulated by both FL-IFX and CONV; however, oncostatin-M [OSM] and IL-6 were reduced more significantly by FL-IFX [both p < 0.001], while IL-17A was only decreased by CONV [p = 0.080]. [D] Proteins in the cell cycle and apoptosis pathway were reduced more significantly by FL-IFX than by CONV; both TGF-α and caspase-8 [CASP-8] were reduced more significantly by FL-IFX [p < 0.001 and 0.007, respectively]. [E] Proteins in the tissue remodelling pathway were significantly regulated by both FL-IFX and CONV, although hepatocyte growth factor [HGF] was reduced more significantly by FL-IFX [p < 0.001]; the concentration of matrix metalloproteinase-1 [MMP-1] was comparable between groups [p = 0.089]. A value of p < 0.05 was considered significant for all tests with false discovery rate [FDR] correction based on the Benjamini–Hochberg procedure.

Figure 3.

Patients within the conventional [CONV] treatment group treated with exclusive enteral nutrition [EEN] or oral prednisolone show similar immune signatures at 10–14 weeks. [A] Fold changes in the median protein concentrations of all 83 proteins between baseline and 10–14 weeks for patients who received EEN or prednisolone treatment; Wilcoxon signed-rank test. [B] Volcano plot of median protein concentrations at 10–14 weeks for EEN-treated patients [n = 18] compared to oral prednisolone-treated patients [n = 14]. None of the 83 proteins with |log₂FC| < 0.5 were significantly different after FDR correction; Mann–Whitney U-test. [C] The concentration of interleukin [IL]-7 was higher in prednisolone patients while the concentration of adenosine deaminase [ADA] was lower in prednisolone- compared to EEN-treated patients at weeks 10–14; binary logistic regression analysis. [D] After FDR correction, the concentrations of IL-7 and ADA did not reduce significantly between baseline and after 10–14 weeks of induction treatment in either EEN- or prednisolone-treated patients; Wilcoxon signed-rank test; p < 0.05 was considered significant for all tests.

Figure 4.

Hierarchical clustering based on the baseline profiles of the 30 IFX-modulated proteins reveals two clusters of patients. [A] Heatmap of the Spearman’s correlation coefficient values between various clinical characteristics of CD and the serum concentrations of the 30 FL-IFX-modulated proteins at baseline [unpaired samples, n = 85]; wPCDAI, weighted paediatric Crohn’s disease activity score; ESR, erythrocyte sedimentation rate [mm/h]; CRP, C-reactive protein [mg/L]; modified GHAS, modified global histological activity score; activity GHAS, activity global histological activity score; SES-CD; simple endoscopic score for Crohn’s disease; neutrophil Cx., neutrophil concentrations [10⁹/L]; leukocyte Cx., leukocyte concentrations [10⁹/L]; F. calprotectin, faecal calprotectin [µg/mL]; albumin, serum albumin [g/L]. [B] Heatmap with hierarchical clustering [Euclidean distance, Ward method] of the 30 IFX-modulated proteins at baseline [paired samples, n = 78] revealed two patient clusters: CD-hi, with a high inflammation profile, and CD-lo, with a lower inflammation profile. The proportions of patients receiving first-line infliximab [FL-IFX] and conventional treatment [CONV] were not significantly different between clusters [p = 0.535]. [C] Volcano plot showing the serum concentrations of 23 of the 30 FL-IFX-modulated proteins were significantly higher in the CD-hi cluster than CD-lo cluster at baseline [unpaired samples, n = 85; Mann–Whitney U-test]. [D] Comparison of the fold changes in the median serum concentrations of the 30 FL-IFX-modulated proteins between the CD-hi and CD-lo clusters after induction treatment. A value of p < 0.05 was considered significant for all tests.

Figure 5.

CD-hi patients have significantly higher median clinical disease activity, C-reactive protein and neutrophil concentrations. [A] Violin plots of the top four most differently expressed proteins in the CD-hi and CD-lo clusters at baseline [unpaired samples, n = 85]. Oncostatin-M [OSM]; tumour necrosis factor ligand superfamily member 14 [TNFSF14]; hepatocyte growth factor [HGF]; transforming growth factor alpha [TGF-α], ***p < 0.001 after false discovery rate [FDR] correction. [B–L] Comparison of clinical disease parameters between the CD-hi and CD-lo clusters at baseline [unpaired samples, n = 85]. [B] Activity GHAS, activity global histological activity score; [C] modified GHAS; [D] wPCDAI, weighted paediatric Crohn’s disease activity score; [E] SES-CD, simple endoscopic score for Crohn’s disease; [F] CRP, C-reactive protein [mg/L]; [G] ESR, erythrocyte sedimentation rate [mm/h]; [H] faecal calprotectin [µg/mL]; [I] serum albumin [g/L]; [J] neutrophil concentration [10⁹/L]; [K] leukocyte concentration [10⁹/L]; [L] thrombocyte count [10⁹/L]. Mann–Whitney U-test; **p < 0.05 after FDR correction; *p < 0.05 before FDR correction; ns, no significant difference.

Figure 6.

Baseline serum immune profiles are associated with clinical remission without treatment escalation at week 52. [A] Barplot with proportion of patients in the CD-hi cluster [high baseline inflammation profile] and CD-lo cluster [lower baseline inflammation profile] achieving clinical disease remission without treatment escalation at week 52 after FL-IFX or CONV treatment. Each bar is the proportion out of 100%. [B] Multivariate binary logistic regression analysis of clinical disease remission without treatment escalation at week 52. [C] Barplot with proportion of patients with moderate [>40–57.5] and severe [>57.5] weighted paediatric Crohn’s disease index [wPCDAI] scores at baseline achieving clinical disease remission without treatment escalation at week 52 after FL-IFX or CONV treatment. Each bar is the proportion out of 100%. [D] Multivariate binary logistic regression analysis of clinical disease remission without treatment escalation at week 52. [E] Barplot with proportion of patients in the CD-hi cluster and CD-lo cluster receiving additional treatment before week 52 after induction treatment with FL-IFX or CONV. Each bar is the proportion out of 100%. [F] Multivariate binary logistic regression analysis of patients who required treatment escalation within 52 weeks. A value of p < 0.05 was considered significant weeks. [G] Barplot of proportion of patients with a moderate wPCDAI [MD, 40–57.5] and severe wPCDAI [SD, >57.5] receiving additional treatment before week 52 after induction treatment with FL-IFX or CONV. Each bar is the proportion out of 100%. [H] Multivariate binary logistic regression analysis of patients who required treatment escalation within 52 weeks. [I, J] Kaplan–Meier analyses of treatment escalation in weeks [unpaired samples, n = 85; log-rank-test] stratified [E] by treatment group, CD-lo/CD-hi cluster and moderate/severe wPCDAI, and [F] by CD-lo/CD-hi cluster and moderate/severe wPCDAI.

Figure 7.

Hierarchical clusters based on IFX-modulated proteins at 10–14 weeks are not associated with clinical remission without treatment escalation at week 52. [A] Heatmap with hierarchical clustering [Euclidean distance, Ward method] of the 30 IFX-modulated proteins at 10–14 weeks [paired samples, n = 78] revealed two clusters: CD#1 10–14 weeks [high inflammation profile] and CD#2 10–14 weeks [low inflammation profile]. The proportion of patients in the CD-lo [lower baseline inflammation profile] and CD-hi [high baseline inflammation profile] clusters [see Figure 4] was not significantly different between the CD#1 and CD#2 10–14 weeks clusters; however, the CD#2 10–14 weeks cluster contained a higher proportion of patients treated with FL-IFX than the CD#1 10–14 weeks cluster [p < 0.001]. [B] Barplot with the proportion of patients treated with FL-IFX or CONV in the CD#1 and CD#2 10–14 weeks cluster achieving clinical disease remission without treatment escalation at week 52. Each bar is the proportion out of 100%. [C] Multivariate binary logistic regression analysis of clinical disease remission without treatment escalation at week 52. [D] Barplot with the proportion of patients treated with FL-IFX or CONV in the CD#1 and CD#2 10–14 weeks cluster receiving additional treatment before week 52 after induction. Each bar is the proportion out of 100%. [E] Multivariate binary logistic regression analysis of treatment escalation at week 52. [F] Barplot with the proportion of patients treated with FL-IFX or CONV in the CD#1 and CD#2 10–14 weeks cluster [re]starting with anti-TNF within 52 weeks. Each bar is the proportion out of 100%. [G] Multivariate binary logistic regression analysis of patients [re]starting anti-TNF within 52 weeks; p < 0.05 was considered significant for all tests. FL-IFX, first-line infliximab; CONV, conventional induction therapy.