Clinical Use and Mechanisms of Infliximab Treatment on Inflammatory Bowel Disease: A Recent Update

The pathogenesis and treatment of inflammatory bowel disease (IBD) have been recently advanced, while it is still challenged with high morbidity and poor prognosis. Infliximab, a monoclonal antibody of tumor necrosis factor (TNF), has emerged as an efficient treatment with many clinical benefits such as quick disease activity reduction and IBD patient life quality improvement. However, the biological effects of infliximab on IBD need to be elucidated. This paper reviewed the clinical use and recently advanced biological action of infliximab on IBD. By forming the stable complex with the soluble or the membrane form of TNF in fluid environment or on cell surface of immune cell, fibroblast, endothelium, and epithelium, infliximab quenches TNF activity and performs the important biological actions which lead to amelioration and remission of immune responses. The mechanisms of infliximab treatment for IBD were intensively discussed. The recent advances on two topics including predictors and side effects of infliximab treatment were also reviewed.


Introduction
In�ammatory bowel disease (IBD), mainly containing Crohn's disease (CD) and ulcerative colitis (UC), is a group of chronic in�ammatory disorders in the colon and small intestine. Although the etiology of IBD remains unclear, the pathogenesis of IBD has been recently advanced. It is strongly suggested that altered immunological function, resulting from an interplay between genetic susceptibility and certain environmental factors including bacteria infection, contributes to the development of mucosal in�ammatory responses of gastrointestinal tract [1]. Proin�ammatory cytokines, especially tumor necrosis factor (TNF), are produced mainly by activated immune cells in in�amed mucosa during the process of IBD, and those proin�ammatory cytokines further activate immune cells, as the feedback, to produce toxic molecules including super oxygen products, chemokines, proteinases, and cytokines which result in tissue damage and in�ammation development [2,3].
In the past years, TNF has been known to play a pivotal role in the pathogenesis of IBD [4]. When released by active macrophages and T lymphocytes, TNF initiates multiple biological reactions below: modulates immune cell function, drives adaptive immune responses, triggers epithelium apoptosis and breaks epithelial barrier, induces endothelium expressing adhesion molecules such as intercellular adhesion molecule 1 (ICAM1) to recruit immune cells, and regulates matrix metalloproteinase (MMP) expression to induce tissue degradation and damage [5,6]. Clinical studies have shown that TNF protein and mRNA levels are elevated in serum, intestinal tissue, stool of active IBD, in correlation with disease activity [4,[7][8][9]. Clinical inhibition of TNF production has been linked with disease remission, improved life quality, and relapse prevention, meanwhile, failure of clinical treatment of IBD has been attributed to early reactivation of TNF secretory capacity by immune cells [10,11]. e �ndings suggest that TNF is critical for disease development. Inhibiting TNF production in in�amed mucosa is one of the important goals for IBD management.
e conventional treatments of IBD include corticosteroids and aminosalicylates. However, only 50% of patients achieve sustained remission with the conventional drugs which can raise many side effects [12]. Recently, many novel drugs have been developed for clinical IBD management, and among them, TNF neutralization by monoclonal antibodies has been shown as one of the effective approaches for IBD treatment [13].
TNF is primarily a type II transmembrane protein with 212 amino acid sequence and exists in a stable homotrimer. TNF alpha converting enzyme (TACE) is a metalloprotease which can cleave membraneintegrated TNF and release TNF in a soluble homotrimeric form [4,9]. Both membrane and soluble TNF can perform their biological function by binding to their receptors including receptor 1 (TNFR1, CD120a) and TNFR2 (CD120b), which are expressed by most tissue cells. rough its receptor TNFR1, TNF can activate intracellular pathways including transcription factor nuclear factor-B (NF-B) and mitogenactivated protein kinase (MAPK), which are key kinases in regulating immune cell activation and migration, inducing gene expression of chemokines, cytokines, and toxic molecules including reactive oxygen species [4,9].
e technique of TNF blockade by monoclonal antibodies has been developed as an effective therapy for immune diseases including IBD [14,15]. As the �rst monoclonal TNF antibody approved for human treatment, in�iximab is a puri�ed, recombinant DNA-derived chimeric humanmouse IgG monoclonal antibody and contains murine heavy (H) and light (L) chain variable regions (VH and VL, resp.), ligated to genomic human heavy and light chain constant regions [14,16]. In�iximab can quickly form stable complexes with the human soluble or the membrane form of TNF and terminate the biological activity and signals of TNF [11]. With a serum half-life of 9.5 days and still detectable in serum of IBD patients 8 weeks aer infusion treatment, in�iximab provides a useful strategy to neutralize TNF and to inhibit immune responses of IBD [17]. Aer approved by the Food and Drug Administration of USA (FDA) in 1998, in�iximab has been used for IBD treatment for about 15 years, and its clinical efficacy has been well studied.
At the beginning of clinical application, in�iximab had been used for Crohn's disease patients with intestine in�ammation. Targan et al. conducted a 12-week multicenter, double-blind, placebo-controlled trial of cA2 (in�iximab) in Crohn's disease patients [50]. 108 moderate-to-severe patients resistant to steroid randomly conducted a single two-hour intravenous infusion of either placebo or cA2 in a dose of 5 mg/kg body weight, 10 mg/kg, or 20 mg/kg. At four weeks, 22 of 27 patients given 5 mg/kg, 14 of 28 given 10 mg/kg, and 18 of 28 given 20 mg/kg had had a clinical response, as compared with 4 of 24 patients in the placebo group ( for the comparison of the cA2 group as a whole with placebo). 33% patients given cA2 went into remission, as compared with 4% of the patients given placebo ( . At 12 weeks, 34 of 83 cA2-treated patients had had a clinical response, as compared with 3 of 25 patients in the placebo group ( 8 . In�iximab for Crohn's disease remission maintenance was also reported [51].
Besides clinical use for intestinal in�ammation, in�iximab has currently been authorized for another two phenotypes of active Crohn's disease: stricturing disease (which causes narrowing of the bowel) and penetrating disease (which causes �stulae or abnormal connections of the bowel) [52][53][54]. In a multicenter, double-blind, randomized, placebo-controlled trial [55], 306 Crohn's disease patients with one or more draining abdominal or perianal �stulas of at least three months' duration received in�iximab (5 mg/kg) intravenously on weeks 0, 2, and 6. A total of 195 patients who had a response at weeks 10 and 14 and 87 patients who had no response were then randomly assigned to receive placebo or in�iximab every eight weeks and to be followed to week 54. e time to loss of response was signi�cantly longer for patients who received in�iximab maintenance therapy than for those who received placebo maintenance (more than 40 weeks versus 14 weeks, ). At week 54, 19 percent of patients in the placebo maintenance group had a complete absence of draining �stulas, as compared with 36 percent of patients in the in�iximab maintenance group ( . As shown by this and other trials, in�iximab was effective in short-term closure of rectovaginal �stulas and maintenance treatment was more effective than placebo in prolonging rectovaginal �stula closure. Clinical efficacy of in�iximab on ulcerative colitis has been clari�ed. Many clinical trials evaluated the effect of in�iximab as induction and maintenance therapy for the patients with ulcerative colitis. In two randomized, doubleblind, placebo-controlled studies -the Active Ulcerative Colitis Trials 1 and 2 (ACT 1 and ACT 2, resp.,) [56], 364 patients with moderate-to-severe active ulcerative colitis were given placebo or in�iximab (5 mg or 10 mg/kg) intravenously at weeks 0, 2, and 6 and then every eight weeks through week 46 (in ACT 1) or week 22 (in ACT 2). In ACT 1, 69 percent of patients who received 5 mg of in�iximab and 61 percent of those who received 10 mg had a clinical response at week 8, as compared with 37 percent of those who received placebo ( for both comparisons with placebo). In ACT 2, 64 percent of patients who received 5 mg of in�iximab and 69 percent of those who received 10 mg had a clinical response at week 8, as compared with 29 percent of those who received placebo ( for both comparisons with placebo). In both studies, patients who received in�iximab were more likely to have a clinical response at week 30 ( for all comparisons). In ACT 1, more patients who received 5 mg or 10 mg of in�iximab had a clinical response at week 54 (45 percent and 44 percent, resp.,) than did those who received placebo (20 percent, for both comparisons). Recently, other studies further showed that in�iximab is an alternative treatment for induction and maintenance therapy in moderately to severely active ulcerative colitis [57].
In soluble or transmembrane form, TNF plays multiple functions in the process of IBD. By blocking and neutralizing TNF activity, in�iximab has shown its high effectiveness in the clinical management of Crohn's disease and ulcerative colitis. �p to now, biological action of in�iximab on IBD has been explored (Table 1).

3.�. In�i�iting In�ammato�� C�to�ines and Mediato�s.
During the process of IBD, TNF is highly produced by immune cells, and as the feedback, TNF recruits immune cells to move from blood vessel into in�ammation sites and induces the cells to release in�ammatory cytokines and mediators [58,59]. Inhibiting TNF production by in�iximab can downregulate the expression of in�ammatory cytokines and mediators in intestinal tissues and ameliorate IBD [18,19]. Ljung et al. studied the time-course of the effects of in�iximab with reference to mucosal cytokine and inducible nitric oxide synthase expression [20]. irty-two patients with Crohn's disease were involved in in�iximab treatment, and clinical response was seen in 14 patients with downregulation of global immunohistochemistry expression, reaching nadir day 3. Immunohistochemical staining showed that rectal nitric oxide was increased at baseline in patients compared with control people. Aer 28 days treatment, decreased rectal nitric oxide levels parallel downregulation of inducible nitric oxide synthase, TNF, IFN-, and IL-1 levels in the patients with clinical response. Olsen and other researchers found that in�iximab therapy decreased the levels of TNF and IFN-mRNA in colonic mucosa of ulcerative colitis patients [60]. irty-two patients with active ulcerative colitis received in�iximab therapy showed clinical and endoscopic improvements. In�iximab reduced the expression of TNF and IFN-mRNA, but not that of IL-10 and IL-4 mRNA in mucosa of ulcerative colitis patients. Reductions in TNF mRNA were correlated to clinical and endoscopic improvements.
Granulocyte-macrophagecolony-stimulating factor (GM-CSF) is linked with the pathogenesis of IBD [61]. Agnholt and colleagues studied the effect of in�iximab treatment on GM-CSF production in Crohn's disease patients [21]. Biopsies from Crohn's disease patients before and aer in�iximab administration and from ten healthy subjects were cultured, and GM-CSF content was analyzed aer 5 days. GM-CSF production was increased in Crohn's disease patients compared with healthy controls and correlated with Crohn's disease activity index. GM-CSF levels and mucosal histology score were decreased aer three in�iximab infusions, indicating the pivotal role of in�iximab treatment in controlling GM-CSF production, in�ammation, and disease activity in Crohn's disease.

Modulating Immune Cell
Functions. Excessive activation of innate and adaptive immune cells contributes to persistent development of IBD [62,63]. Moreover, mucosal T cells show resistance to activation-induced apoptosis in Crohn's disease, leading to accumulation of T cells in mucosa and large amount of cytokines production [64]. One action of in�iximab treatment is to induce immune cell apoptosis by outside-to-inside signaling through transmembrane TNF (mTNF). Van den Brande et al. cultured peripheral blood lymphocytes and lamina propria T cells from patients with Crohn's disease or from healthy volunteers and treated the cells with in�iximab or etanercept, two TNF-neutralizing drugs, respectively [22]. By using �uorescence-activated cell sorter analysis, annexin V staining, and cleaved caspase-3 immunoblotting, they found that in�iximab bound to peripheral blood lymphocytes and lamina propria T cells from patients with Crohn's disease, and subsequently induced apoptosis of activated lymphocytes, compared with those from healthy volunteers, whereas etanercept did not show the same effect. Other studies had been performed to verify the in vivo effect of in�iximab on mucosal T cell death from Crohn's disease. In a pilot study, 10 Crohn's disease patients received three consecutive infusions of in�iximab, endoscopic intestinal biopsies were collected aer 10 weeks of treatment. As detected by T�NEL assay, in�iximab treatment induced a sustained mucosal T apoptosis in Crohn's disease patients [23]. Another group also found that infusion of in�iximab in steroid refractory patients with Crohn's disease induced a signi�cant increase in CD3 and T�NEL positive cells within colonic biopsies, and those in�iximab-stimulated apoptotic cells were activated T lymphocytes but not resting cells [24].
IBD is characterized by imbalance T helper cell () responses, with dominance of 1 and 17 responses while de�ciency of 2 and Treg responses [65][66][67]. Downregulating the established 1 cell responses, or shiing to 2 and Treg responses, can successfully ameliorate IBD, as indicated by clinical and experimental research [25][26][27]. Agnholt and Kalto studied in vitro the effect of in�iximab on lamina propria T cell responses [28]. Mucosal T cell cultures from patients with Crohn's disease produced signi�cantly higher levels of IFN-and TNF than T cell cultures from healthy controls, while the production of IFN-was downregulated by in�iximab treatment in early T cell cultures from Crohn's disease patients. Matsumura et al. have recently shown that 2 weeks in�iximab infusion therapy can also modulate 1 and 2 balance of active ulcerative colitis and induce remission of the ulcerative colitis [68]. In�iximab therapy has been recently shown that it can modulate regulatory T cell (Treg) response in mucosal of IBD patients. Inappropriate immune responses including dominant 1 and 17, while de�cient Treg contributes to the progress of the intestinal immune system in IBD. Increased apoptosis of Treg was detected in IBD, suggesting that a numerical de�ciency of Treg responds to an insu�cient compensation of chronically activated T lymphocytes [29]. Recent studies have been reported that in�iximab treatment rapidly increases the frequency of functional Treg in active IBD, affects Treg activation and expansion, and enhances the suppressive function of Treg [29,30].
Regulatory macrophage is a subtype innate immune cell with anti-in�ammatory properties, appears to modulate in�ammatory immune responses, and plays an important role in wound healing and gut homeostasis [31]. e effect of in�iximab treatment on regulatory macrophage induction has recently been explored. Hommes and colleagues have shown that the antibodies against TNF including in�iximab and adalimumab induce formation of regulatory macrophages in an Fc region-dependent manner. e induced regulatory macrophages inhibit proliferation of activated T cells, produce anti-in�ammatory cytokines, and express the regulatory macrophage marker CD206 [31].

Protecting Intestinal Epithelial Cells from Apoptosis.
Loss of epithelial barrier integrity, characterized with increased apoptosis of epithelia, is considered an early step in the pathogenesis of IBD [32]. TNF, as the major proin�ammatory mediators in IBD, is one of the extrinsic signals which initiate apoptosis of enterocytes [69]. e action of in�iximab treatment is linked with protecting intestinal epithelial cells from apoptosis. A clinical study presented that epithelial apoptoses were upregulated in the colon in Crohn's disease and restored to normal in majority of patients by in�iximab treatment [33], which correlated with epithelial barrier dysfunction measured as epithelial resistance.
Enterocyte apoptosis contributes to intestinal barrier dysfunction and initiation of intestinal in�ammation [70]. Suenaert and colleagues studied intestinal permeability in refractory Crohn's disease before and aer treatment with in�iximab [32]. 4 weeks in�iximab infusion decreased intestinal permeability of active Crohn's disease, measured by urinary excretion of [32] Cr-EDTA aer oral intake, con�rming the essential role of TNF in modulating gut barrier in IBD.

Restoring Endothelium Function and Blocking Angiogenesis.
Endothelium has multiple functions such as forming the semiselective barrier between the vessel lumen and surrounding tissue, controlling the passage of materials and in�ltration of imune cells into tissues. During the process of IBD, the expressions of leukocyte/endothelial cell adhesion molecules such as intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) were upregulated, which recruit leukocyte migration into intestinal tissue, as shown by the Affymetrix gene expression microarray study [34]. Aer in�iximab infusion, the upregulated leukocyte cell adhesion molecules were restored, in parallel with the disappearance of the in�ammatory cells from the colonic lamina propria. Also, endothelial cell adhesion molecules and most chemokines/chemokine receptors in colon tissues returned to normal aer in�iximab therapy. e �ndings support the concept that in�iximab regulates immune cell in�ltration by controlling leukocyte/endothelial cell adhesion molecules and chemokines/chemokine receptors.
Endothelial dysfunction was reported in patients with IBD, and attributed to TNF produced in intestine. Schinzari et al. found that endothelium-dependent vasodilation to acetylcholine was impaired in Crohn's disease, but not in ulcerative colitis. TNF neutralizing treatment with in�iximab enhanced the responsiveness of endothelium to acetylcholine in Crohn's disease patients, indicating that endothelial function is impaired in Crohn's disease and is bene�cially restored by in�iximab treatment [35].
Angiogenesis has been shown a critical component of neoplastic and chronic in�ammatory disorders. Recently, local microvasculature was reported to be triggered by vascular endothelial growth factor A (VEGF-A) [36], and feedback to undergo an intense process of in�ammationdependent angiogenesis, and contribute to IBD pathogenesis [71]. Scaldaferri et al. have recently reported that the expression of proliferation marker Ki-67 in endothelial cells was signi�cantly reduced by in�iximab administration, in concomitance with decreased mucosal concentration of VEGF-A [36]. e data indicate another novel effect of in�iximab treatment in downregulating mucosal angiogenesis in IBD and restraining production of VEGF-A.

Regulating the Balance between Matrix Metalloproteinases (MMPs) and Tissue Inhibitor of Metalloproteinases (TIMPs).
Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of MMPs (TIMPs), are produced in the gastrointestinal tract by several structural cells, including neutrophils, platelets, mesenchymal cells, T cells, monocytes, macrophages, and cancer cells [72]. e balance between MMPs and TIMPs is essential for many physiological processes in the gut. However, imbalance between MMPs and TIMPs plays an important role in the pathophysiology of diverse intestinal in�ammatory conditions including IBD [73]. In�iximab treatment can restore the balance between MMPs and TIMP seen in IBD.
Meijer et al. determined expression and secretion of MMP-1, -2, -3, -9, and their inhibitors TIMP-1, -2 by IBD versus control intestinal mucosa ex vivo and to assess the regulatory capacity by in�iximab of the proteolytic phenotype [37]. Intestinal mucosal explants from 20 IBD and 15 control patients were cultured with or without in�iximab and/or the T-cell activator pokeweed mitogen (PWM). Expression of MMP and TIMP protein/activity in basal medium was higher in IBD versus control explants. in�iximab downregulated MMP-1, -3, and -9 relative to TIMP-1 and -2 and also decreased MMP-1 and -3 activities, while PWM enhanced these levels, partly counteracted again by in�iximab. e expression of MMP-2 relative to TIMP did not change by treatment with in�iximab and/or PWM.
Di Sabatino et al. investigated the action of in�iximab on apoptosis, the production of matrix metalloproteinases BioMed Research International 5 (MMPs) and tissue inhibitor of metalloproteinases (TIMPs)-1, and migration of Crohn's disease myo�broblasts [38]. Colonic myo�broblasts were isolated from patients with active Crohn's disease and controls, and treated by in�iximab. Crohn's disease myo�broblasts showed higher mTNF expression than control myo�broblasts. In�iximab had no effect on Crohn's disease myo�broblast apoptosis, caspase-3 activation, and production of MMP-3 and MMP-12. However, in�iximab induced a signi�cant dose-dependent increase in TIMP-1 production. e migration of Crohn's disease myo�broblasts was enhanced signi�cantly by in�iximab and recombinant human TIMP-1, and in�iximab-induced migration was inhibited by anti-TIMP-1 neutralizing antibody. In�iximab also decreased Crohn's disease myo�broblast collagen production. Together, in�iximab therapy can enhance TIMP-1 production and myo�broblast migration, while reduce MMP activity and facilitate the wound healing.
3.6. Improving Mucosal Healing. Mucosal healing has appeared as an important treatment goal for patients with IBD, and can alter the course of IBD with sustained clinical remission and reduced rates of hospitalization and surgical resection [74]. Recently, in�iximab treatment has emerged as a therapy for mucosal healing induction. Kierkus et al. have recently evaluated the impact of in�iximab infusion on induction of intestinal mucosal healing [39]. 66 pediatric patients with Crohn's disease were involved in the study with in�iximab infusion. Healing induction therapy with in�iximab was found to be clinically effective in 72% of pediatric patients with Crohn's disease and induced a remission in 33% of them. Also, induction therapy with in�iximab helps to increase body mass index.
e possible mechanism of in�iximab treatment improving mucosal healing has recently been studied. Kierkus et al. showed that a signi�cant induction of regulatory macrophages was observed in patients with mucosal healing a�er treatment with in�iximab� while induction was absent in patients without mucosal healing. As shown in an in vitro model, the induced regulatory macrophages displayed the ability to induce wound healing, suggesting a key role for in�iximab-induced regulatory macrophages in mucosal healing [39].

3.�. Mo�ula�ing In�amma�or� �ignal �a���a�s.
Nuclear transcription factor-B (NF-B) is identi�ed as the key regulator in immunological setting of IBD [75]. NF-B activation is markedly induced in IBD patients and through its ability to promote the expression of various proin�ammatory genes, NF-B strongly in�uences the course of mucosal in�ammation [76,77]. TNF is one of the best-characterized agonists of NF-B pathway and is itself regulated by NF-B [75]. Turning down NF-B signals is another bioactivity of in�iximab. �uidi et al. evaluated the production of TNF by peripheral blood mononuclear cells and the levels of NF-B family molecules in the intestinal mucosa during in�iximab therapy in 12 patients [40]. In�iximab treatment increased NF-aB inhibitor levels including I B and I B , and thus downregulated NF-B signals and TNF production in the intestinal mucosa of Crohn's disease patients [40].
However, another group found that in�iximab enhances the activity and expression of mitogen-activated protein kinases (MAPKs) during the process of IBD. MAPKs are key signal pathways that cooperate in the orchestration of in�ammatory responses, and extensive cross-talk to other in�ammatory pathways, such as NF-B and Janus kinase/STAT signaling [78]. Four groups of MAPKs have been identi�ed in mammalian cells: the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs) or stressactivated protein kinases (SAPKs), the p38 kinases, and ERK5/big MAPK [79,80]. Waetzig et al. studied the activity and expression of the p38 MAPK alpha-delta, JNKs, and ERK1/2 in the in�amed intestinal mucosa of IBD patients [41]. Western blot analysis revealed that p38 alpha, JNKs, and ERK1/2 were signi�cantly activated in IBD, with p38 alpha showing the most pronounced increase in kinase activity. In vivo inhibition of TNF by in�iximab infusion resulted in a highly signi�cant transient increase of p38 alpha activity during the �rst 48 h a�er infusion. A signi�cant in�iximabdependent p38 alpha activation was also observed in THP-1 myelomonocytic cells. In human monocytes, in�iximab enhanced TNF gene expression, which could be inhibited by SB 203580.

Genetic and Genomic Predictor of �n�i�ima� �reatment
It was noted by clinical trials for IBD that initial response rate to in�iximab treatment was only about 60%, and approximately 30% of the responders sustaining remission throughout 54 weeks [51]. Recently, the markers that predict response to in�iximab treatment of IBD have been developed, such as clinical parameters including patient characteristics, smoking status and disease phenotype, and biological markers including C-reactive protein and serum TNF levels [81]. However, these markers are not so effective and stable to provide predictive value, and few markers have consistently replicated among clinical studies. For example, young age, smoking, and concomitant immunosuppressive treatment were reported as independent variables favoring short-term response to in�iximab [82,83], but no or reverse association was developed in other studies evaluating smoking and age [84,85]. Development on new marker and factors with predictive value for IBD treatment is critical. �enetic predictors of in�iximab responsiveness become appealing, especially those candidate genes thought to participate in the pathogenesis or susceptibility of IBD. Hlavaty et al. reported that polymorphisms in FasL/Fas system and caspase-9 in�uenced the response to in�iximab in luminal and �stulizing Crohn's disease [86]. In the study, Fas ligand-843 TT genotype was presented exhibiting the strongest association with the nonresponse, while concomitant mercaptopurine/azathioprine therapy was linked with the effect of unfavourable genotypes in luminal Crohn's disease. To date, lots of candidate gene polymorphism was reported as potential predicators of anti-TNF response, including IBD5, TNF, TNF receptor, and NOD2 [81], but few gene markers showed high reproducibility among the studies. In the situation, combinations with different markers including genetic and nongenetic markers might be alternative option. A recent interesting study investigated multiple parameters to determine early response to in�iximab in patients with ulcerative colitis [81]. In the study, disease activity, antineutrophil cytoplasmatic autoantibody (ANCA), and markers of in�ammation were measured during 14 weeks in�iximab infusion, and genotyping for IL23R gene variants was performed as well. Multivariate regression analysis identi�ed high disease activity index before IFX therapy and negative ANCA status as independent positive predictors for response to in�iximab. Homozygous carriers of in�ammatory bowel disease (IBD) risk-increasing IL23R variants were more likely to respond to in�iximab than were homozygous carriers of IBD riskdecreasing IL23R variants.
Genome wide association (GWA) predictors of in�iximab responsiveness have been developed. Recently, 71 con�rmed Crohn's disease susceptibility loci were reported by genome wide meta-analysis [87], and associations of the IBD susceptibility loci and novel �pharmacogenetic� GWAS identi�ed loci with primary nonresponse to anti-TNF alpha in pediatric IBD patients were studied [88]. Dubinsky et al. determined associations of phenotype and genotype with primary nonresponse to in�iximab infusion in both pediatric Crohn's disease and ulcerative colitis patients, and identi�ed genetic associations by testing known IBD susceptibility loci and by performing a GWA for primary nonresponse [88]. ey found that the combination of phenotype and genotype was most predictive of primary nonresponse to in�iximab treatment in pediatric IBD. De�ning GWA predictors of response to in�iximab or other biological anti-TNF treatments may provide promise for clinical utility.

�. �ew �ide ��ects o� �n�i�im�b �re�tment
Over the past 10 years, anti-TNF agents including in�iximab have been emerging as an alternative treatment to overcome the shortcomings of conventional drugs and provide most of IBD patients the improved life quality. However, several side effects associated with in�iximab have been recently reported (Table 2), including acute or delayed infusion reactions, leucopenia, serious infection, antichimeric antibody formation, and increased risk of malignancy [42][43][44][45][46][47]89]. Steenholdt et al. showed that acute severe infusion reactions were strongly associated with development of anti-in�iximab IgG antibody, and the risk was particularly high at the 2nd infusion in retreatment series [42]. In�iximab was also reported to exacerbate clonal -T cell expansion in vivo and induced -T cell proliferation in vitro, indicating the potential risk of developing malignant -T cell lymphomas following anti-TNF-agent therapy [48].
It was also reported that in�iximab treatment for IBD induced other autoimmune disease such as psoriasis. A Spanish group reported a 31-year-old woman with extensive Crohn's disease and perianal lesions developed a cutaneous T 2: Side effects of in�iximab treatment.

Conclusions
TNF, as the key proin�ammatory cytokine during the development of IBD, plays the pivotal role in modulating mucosal immune response, including regulating immune cell proliferation, upregulating adhesion molecule expression, and inducing apoptosis of intestinal epithelial cells. By forming stable complexes with the human soluble or the membrane form of TNF expressed on cell surface, including immune cell, �broblast, and epithelium, in�iximab counteracts the activity of TNF, and plays an important role in IBD treatment. e known mechanism of in�iximab is summarized in Table 1. However, the biological action of in�iximab on immune diseases is far from being well explored. For example, 17 cells have been shown to participate in progress of IBD, but so far no evidence is available about impact of in�iximab on 17 cell function during the process of IBD. In�iximab has been thought to exert its biological functions through blocking TNF, while a recent study found that IL-15 and its soluble receptor might mediate the response to in�iximab in patients with Crohn's disease [90]. Furthermore, in�iximab is also clinically used for other immune diseases including arthritis, in which immune responses are not typically characterized by TNF [91,92], in this way, we believe that in�iximab is supposed to play biological action beyond neutralizing TNF production and counteracting TNF activity.

Con�ict o� �nterests
e authors declare that they have no con�ict of interests.
Authors' Contribution Y. Guo, N. Lu, and A. Bai contributed equally to this work.