Potential role of MALT1 as a candidate biomarker of disease surveillance and treatment response prediction in inflammatory bowel disease patients

Abstract Background Mucosa‐associated lymphoid tissue lymphoma translocation protein 1 (MALT1) regulates adaptive and innate immune responses in several inflammatory disease. However, clinical involvement of MALT1 in inflammatory bowel disease (IBD) patients remains unclear. Hence, this study was intended to investigate the correlation of blood MALT1 with disease activity, inflammation indexes as well as treatment response of IBD patients. Methods Blood MALT1 expression in 100 IBD patients [including 25 active (A)‐Crohn's disease (CD) patients, 25 remission (R)‐CD patients, 25 A‐ulcerative colitis (UC) patients, and 25 R‐UC patients] and 25 health controls (HCs) was detected by reverse transcription‐quantitative polymerase chain reaction; besides, serum tumor necrosis factor‐alpha (TNF‐α) and interleukin‐17A (IL‐17A) in IBD patients were detected by enzyme‐linked immunosorbent assay. Results MALT1 was increased in A‐UC patients than in R‐UC patients (p = 0.038) and in HCs (p < 0.001), and also elevated in A‐CD patients than in R‐CD patients (p = 0.048) and in HCs (p < 0.001). MALT1 was positively related to C‐reactive protein (CRP, p = 0.011), TNF‐α (p = 0.036), IL‐17A (p = 0.023), and Mayo score (p = 0.005) in A‐UC patients, CRP (p = 0.017), erythrocyte sedimentation rate (p = 0.033), TNF‐α (p = 0.004), and Crohn's disease activity index score (p = 0.028) in A‐CD patients. But MALT1 was not correlated with either inflammation indexes or disease activity score in R‐UC and R‐CD patients. MALT1 gradually declined from baseline to W12 in A‐UC and A‐CD patients (both p < 0.001). Moreover, MALT1 at W4 (p = 0.031) and W12 (p = 0.003) in A‐UC patients as well as MALT1 at W12 (p = 0.008) in A‐CD patients associated with clinical response. Conclusion MALT1 serves as a potential biomarker for disease surveillance and treatment response prediction of IBD patients.


| INTRODUC TI ON
Inflammatory bowel disease (IBD), characterized by continuing aberrant immune response, is an idiopathic gut-inflammatory disease with an increasing incidence, which predominantly includes ulcerative colitis (UC) and Crohn's disease (CD). [1][2][3][4][5] In order to alleviate clinical symptoms (including abdominal pain, watery, or bloody diarrhea, etc.), promote intestinal mucosa healing, and reduce disease activity, many medical treatments have been applied in IBD patients, including steroids, immunosuppressants, biological treatments, etc. 6,7 However, IBD is incurable and easy to relapse, which brings treatment-cost burden to patients and their families; moreover, reliable methods to monitor disease progression in IBD patients are still lacking. [8][9][10][11] Therefore, it is necessary to explore novel biomarkers to estimate disease risk and activity as well as treatment response of IBD; subsequently provide individual treatment and improve outcomes of IBD patients.
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is located close to B-cell lymphoma (BCL) 2 on chromosome 18q21 and expressed in multiple cell types. [12][13][14] Based on its scaffold function and proteolytic activity, MALT1 regulates immune reactions via activating several signaling pathways (mainly including nuclear factor-kappa-B (NF-κB) signaling pathway, etc.). 15,16 Moreover, MALT1 induces the differentiation of T-helper (Th) 1 and Th17 cells, which further aggravates many inflammatory and autoimmune diseases, such as psoriasis, rheumatoid arthritis, etc. [17][18][19] Besides, as mentioned above, IBD is a chronic inflammatory disease, which is characterized by abnormal intestinal immune response. 4 Therefore, we speculated that MALT1 might be involved in the etiology and development of IBD. However, there is no clinical research noting that MALT1 can serve as a potential biomarker for evaluation of disease risk and activity of IBD.
Hence, this study detected blood MALT1 expression in IBD patients at baseline, week (W) 4 and W12 after treatment, aiming to investigate the correlation of MALT1 with disease activity, inflammation indexes, and treatment response in IBD patients.

| Subjects
This study consecutively enrolled a total of 100 IBD patients who Crohn's disease activity index (CDAI) score ≥150 points, and R-CD patients were required to have a CDAI score <150 points; A-UC patients were required to have a Mayo score ≥3 points, and R-UC patients were required to have a Mayo score <3 points. 21 The patients were excluded from the study if they had other immune system diseases, severe active infections, hematologic malignancies, or cancers. In addition, another 25 healthy subjects with matched gender and age to IBD patients were recruited as health controls (HCs), who were also excluded if they had immune system diseases, severe active infections, hematologic malignancies, or cancers. The study was approved by Institutional Research Ethics Committee of Gansu Province Hospital of Traditional Chinese Medicine.

| Collection of data and samples
Characteristics of all subjects were recorded after enrollment and examination, which included age, gender, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). Besides, CDAI score was collected from CD patients, and Mayo score was collected from UC patients. The higher CDAI score and Mayo score indicated the more severe disease activity. For sample collection, PB samples of all subjects were obtained after enrollment to separate peripheral blood mononuclear cells (PBMCs) and serum. Besides, for A-CD patients and A-UC patients, PB samples were also obtained at W4 and W12 to isolate PBMCs.

| Assessment of inflammatory cytokine level
Inflammatory cytokines in serum of all IBD patients, including tumor necrosis factor-alpha (TNFα) and interleukin-17A (IL-17A), were detected by enzyme-linked immunosorbent assay (ELISA) using commercial Human ELISA Kits (Bio-Techne China Co., Ltd.). The ELISA procedure was strictly followed the instructions of kits.

| Assessment of MALT1 expression
MALT1 expression in PBMCs was detected by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) assay among all subjects after enrollment, as well as A-CD patients and A-UC patients at W4 and W12 after treatment. In brief, total RNA was extracted using RNeasy Protect Mini Kit (Qiagen). Then reserve transcription was completed by PrimeScript™ RT reagent Kit (Takara). After that, qPCR reaction was finished using SYBR ® Premix DimerEraser™ (Takara). The relative expression was calculated by 2 −ΔΔCt method using GAPDH as the internal reference. The primer sequences were referred to the previous study. 22

| Assessment of clinical response
At W12 after treatment, clinical response was assessed among A-CD patients and A-UC patients. Clinical response of A-CD patients was defined as a CDAI score decrease over 70 points from baseline 21 ; clinical response of A-UC patients was defined as a Mayo score decrease over 30% or 3 points from baseline, and the rectal bleeding subscore decrease at least 1 point or the rectal bleeding subscore of 0-1. 23

| Statistical analysis
Statistical analysis and graph plotting were carried out using SPSS

| Characteristics of IBD patients and HCs
The mean ages of A-CD, R-CD, A-UC, R-UC, and HCs were 31.6 ± 9.8 years, 38

| Expression of MALT1 in IBD patients and HCs
Generally, difference was observed in MALT1 among A-CD patients, R-CD patients, A-UC patients, R-UC patients, and HCs (p < 0.001, Figure 1A). In detail: MALT1 in both A-UC patients and A-CD patients was elevated than that in HCs (both p < 0.001); besides, MALT1 was elevated in A-UC patients versus R-UC patients (p = 0.038) and A-CD patients versus R-CD patients (p = 0.048).  Figure 1C).

| Correlation of MALT1 with disease activity score in IBD patients
MALT1 was positively correlated with mayo score in A-UC patients (r = 0.541, p = 0.005), but they were not correlated in R-UC patients (r = 0.357, p = 0.080) (Figure 2A,B). In terms of CD, MALT1 was positively associated with CDAI score in A-CD   Table 2).

| DISCUSS ION
Although the original of MALT1 remains unclear, the current studies speculate that MALT1 might originate from mucosaassociated lymphoid tissue. 24 Additionally, MALT1, together with its caspase recruitment domain (CARD) family member BCL10, activates NF-κB signaling pathway and regulates innate and adaptive immunoreaction. [25][26][27] Furthermore, aberrant expression of MALT1 is linked with immunodeficiency. 28,29 In view of the fact that IBD patients are usually along with immunodeficiency; consequently, MALT1 may be dysregulated in IBD patients. 30,31 However, no relevant study investigates MALT1 expression in IBD patients. In this study, we found that MALT1 was elevated in IBD patients than HCs; additionally, MALT1 had good value to distinguish IBD patients from Several lines of evidence support that MALT1 regulates inflammation level in some autoimmune diseases, such as psoriasis, ankylosing spondylitis, rheumatoid arthritis, etc. [36][37][38][39] For instance, one study discloses that the inhibition of MALT1 suppresses the inflammatory response in proteoglycan-induced ankylosing spondylitis mouse models. 37 Considering that the autoimmune disease shared similar etiopathogenesis to some extent, meanwhile T-cell activation and further leading to the production of various inflammatory cytokines commonly occurred in these patients, we hypothesized that MALT1 might be related to inflammation in IBD patients. [40][41][42] In this study, we found that MALT1 was positively correlated with inflammation indexes and disease activity score of active IBD patients. The reason might be as follows: (a) MALT1 activated NF-κB signaling pathway; meanwhile the activation of NF-κB pathway was  Apart from what mentioned above, we also disclosed that MALT1

MALT1 expression in A-CD
in both A-CD and A-UC patients was gradually declined after treatment. Of note, MALT1 at W4 and W12 in A-UC patients as well as MALT1 at W12 in A-CD patients associated with clinical response.
The possible reasons might be that: (a) As described, MALT1 expression indicated the inflammation level in IBD patients, whose inflam-

| CON CLUS IONS
In general, MALT1 is abnormal expressed and related to aggravated disease activity score, inflammation indexes as well as treatment response in IBD patients. Hence, MALT1 may serve as a potential biomarker for disease surveillance and treatment outcome prediction of IBD.

ACK N OWLED G EM ENTS
This study was supported by The Science and Technology Project of Yulin City (YF-2020-052).

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.