Serum biomarkers
C-reactive protein (CRP)
CRP is one of the most ubiquitously used biomarkers given its low cost, ease of testing, and well-established protocols regarding its usage. It is one of the body’s acute-phase reactants, and its production is stimulated in hepatocytes by pro-inflammatory cytokines.18,19 Its utility as an indicator of inflammation is related to its relatively short half-life of 19 hours compared to other acute-phase proteins.10 Though CRP is widely used as a biomarker for IBD, its diagnostic value is limited by its lack of specificity. Elevations in CRP can also be caused by other inflammatory conditions such as autoimmune disorders, infections, and malignancies.19 Thus, CRP levels cannot be diagnostic of IBD in isolation but must be interpreted along with the clinical picture.20
In the absence of inflammation, serum CRP is typically low (< 1 mg/L) but can increase over 1,000-fold in the setting of acute inflammation.9 Prior studies on CD have found a significant association between CRP elevation and moderate to severe clinical activity and evidence of active disease on ileocolonoscopy. However, due to unclear causes, there has not been a strong correlation between CRP levels and disease activity in UC.10,21 Possible explanations include the difference in IL-6 production in UC and CRP production by mesenteric adipocytes in patients with CD.22
Conversely, normal CRP does not rule out active IBD.11 Prior studies have found a subset of patients with Crohn’s disease harboring genetic variations that limit CRP elevations.23 Based on the ACCENT1 trial, patients with elevated baseline CRP and those whose CRP normalized by week 14 of treatment with infliximab were more likely to maintain clinical remission and treatment response. Thus, CRP can be a useful biomarker24 in those whose CRP levels correspond to their disease activity.
Recent guidelines for UC management suggest monitoring CRP and fecal calprotectin in asymptomatic individuals to avoid more costly and invasive testing, such as endoscopy, for routine disease activity assessment.25 Similar guidelines were released following the CALM study, which showed improved clinical and endoscopic outcomes in patients with CD when therapy decisions were based on clinical symptoms and biomarkers rather than symptoms alone.26 A large prospective observational study of CD patients in a tertiary referral center showed that asymptomatic patients with elevated CRP levels were over twice as likely to be hospitalized over a two-year follow-up period.27 This study provides real-world evidence that CRP is a useful biomarker for predicting clinical outcomes in CD patients.
Salivary CRP also presents an intriguing alternative to typical serum sampling as an even more easily obtainable biomarker of IBD activity for disease tracking in select patients. Future studies are needed to establish optimal clinical applications for this alternative to venous sampling.28
ESR
Erythrocyte sedimentation rate (ESR) is commonly tested in conjunction with CRP. It is a measure of inflammation based on how quickly erythrocytes sediment through plasma in a column. A higher sedimentation rate indicates inflammation. Like CRP, elevations in ESR are not specific to IBD and can occur in response to any inflammatory stimulus. It differs from CRP in that it peaks more slowly and takes longer to return to normal. Additionally, it does not show the same variability with UC that CRP does and tends to respond similarly to the inflammation seen in UC and in CD.19
It is important to remember that ESR can be affected by other physiological factors such as pregnancy, age, and gender, as well as changes in hematocrit seen in patients with anemia and polycythemia.19 Additionally, changes in the size of erythrocytes can also affect ESR values, such as those seen in certain disease states or as a side effect of some medications.29 This becomes particularly important when monitoring ESR in patients on azathioprine or 6-mercaptopurine, as these medications have been shown to cause elevated ESR despite normal CRP and no clinical evidence of active disease.30
Vitamin D
Vitamin D is an immune modulator involved in both innate and adaptive immunity. It is primarily produced in the skin upon exposure to sunlight (UVB) or absorbed by the small intestine following food intake.12 Vitamin D deficiency in IBD patients is associated with an increased risk of disease recurrence, hospitalizations, and surgeries.9 Vitamin D deficiency is common in the general population due to inadequate exposure to sunlight, impaired enzymatic activation, lower bioavailability, insufficient physical activity, and smoking. In addition to these common risks, IBD patients have an increased risk of osteopenia and osteoporosis due to malabsorption of calcium and vitamin D caused by disease flares or prior surgery, dietary restrictions, and frequent use of medications that inhibit bone formation or increase bone turnover.29
Serum vitamin D levels are lower in IBD patients compared to those with IBS. In patients with CD, vitamin D levels negatively correlate with disease activity and inflammatory markers such as CRP.12 Multiple studies have even suggested a role for vitamin D deficiency in the pathogenesis of CD, not simply as a consequence of the disease itself. Thus, robust supplementation of vitamin D may be of therapeutic benefit.31 Similar effects have been demonstrated in UC, with vitamin D interacting with anti-inflammatory serum cytokines.32 Patients with active IBD and those who have required over three months of steroid treatment should have their calcium and vitamin D levels monitored. Physicians should have a low threshold to start supplements to prevent low bone mineral density.13 Studies investigating therapeutic effects of vitamin D related to IBD are limited, and further research is needed to determine the optimal range and therapeutic potential of vitamin D.
Platelets
Platelets are a commonly tested lab value yet are often overlooked in the evaluation of IBD patients. Evidence has increasingly shown that, in addition to their primary hemostatic function, platelets also play an active role in multiple inflammatory processes. “Reactive thrombocytosis” is now a well-established phenomenon in the setting of inflammation.33 Many changes in platelet structure and function occur in IBD, especially in the setting of active disease. Inflamed bowel tissue secretes platelet activation factor, which affects circulating platelet levels and coagulation.34 Compared to healthy controls, the platelets of those with IBD are more sensitive to activation, even in clinically silent disease.33 Some small studies have even suggested that increased platelet counts in patients who have UC with mucosal healing could be predictive of relapse.35
Mucosal capillary thrombi have been identified in rectal biopsies of patients with CD and UC, suggesting that platelets may be involved in chronic intestinal inflammation. This finding does not appear to correlate with disease severity or the extent of inflammation in IBD patients, but these microthrombi are consistently absent in the mucosa of normal subjects.33
Fecal biomarkers
Fecal biomarkers, primarily composed of fecal leukocyte proteins, are commonly used to assess disease severity in patients with IBD). They may be preferred over blood samples at times due to ease of sample accessibility and higher specificity for gastrointestinal inflammation.19 Fecal biomarkers are likely to be the most accurate in individuals who have previously manifested an elevation and those whose biomarker activity correlates with endoscopic disease severity.25
However, studies indicate that compliance rates rarely exceed 60% for various reasons36 including forgetfulness, lack of perceived benefit, and reluctance to handle feces.37 Despite being an invasive procedure, blood collection is typically more readily accepted by most patients and can be completed expeditiously during routine follow-up visits. Current studies are underway to assess the viability of home fecal calprotectin tests akin to home testing for diabetes and hypertension, with patient reporting.38 However, further studies supporting the utility and reliability of these tests are needed.
Fecal calprotectin
Calprotectin is released by activated innate immune cells in response to cell damage or stress.29 It belongs to the S100 family of proteins and serves to regulate protein phosphorylation, intracellular calcium regulation, and protection against oxidative cell damage within neutrophils. Its extracellular functions include antimicrobial and antifungal activities, as well as regulation of apoptosis and inflammation.14
Causes of elevated fecal calprotectin (FC) other than IBD include NSAID enteropathy, pancreatic insufficiency, alcoholic enteropathy, colorectal cancer, and microscopic colitis. Since neutrophils are relatively scarce in normal intestinal mucosa, FC levels are low in healthy individuals.39 For this reason, it is also useful for distinguishing between functional and organic diseases, especially in the setting of known IBD who may also have IBS overlap symptoms despite adequate control of inflammation.40 The sensitivity and specificity of FC for Crohn’s disease are 100% and 97%, respectively, compared to IBS.29,40 Fecal calprotectin also has the potential to differentiate between perianal fistulas due to CD and cryptoglandular perianal fistulas,41 which is a common benign anorectal disorder that is mainly managed with surgery.42 A meta-analysis indicated that FC testing could reduce endoscopy by 67% in adults, although it could lead to treatment delays in 6% of patients due to false negatives when used as a screening tool for IBD.29,43
Fecal calprotectin is valuable for assessing active disease and monitoring treatment response, as FC levels decrease with mucosal healing. Persistently high levels in IBD patients in remission could predict a higher risk of disease relapse within the next 12 months.44 This correlation may have a higher predictive value for UC than for CD, likely due to differences in inflammatory patterns.45 In UC, an FC level ≤ 250 µg/g following biologic induction was associated with a higher probability of achieving clinical, endoscopic, and histologic remission by week 52, as well as a decreased probability of colectomy within 7 years.46 Elevated FC in UC patients in clinical and endoscopic remission has also been associated with the risk of relapse.47
Nevertheless, recent studies have shown that FC remains a useful tool for evaluating active disease in isolated small bowel CD with both inflammatory and stenotic disease but may not be as effective for monitoring penetrating disease.48 The combined evaluation of FC, hemoglobin, and CRP at least once may improve CD monitoring and management through risk matrices.49 Notably, FC from ileostomy output demonstrates high sensitivity and specificity for monitoring small bowel inflammation and disease recurrence in post-operative CD patients.50
Establishing baseline FC levels has been challenging due to differences in extraction methods and variable baseline levels among certain populations. For example, individuals from areas with poor sanitation may have elevated baseline FC levels.11 Children also have a lower reference range for FC than adults.39 There may also be variability depending on the time of sample collection throughout the day. Therefore, it is generally recommended to collect samples in the morning to standardize testing and reduce variability.51,52 Despite these variations, most studies agree that FC levels of 150–250 µg/g indicate active disease.11
Fecal lactoferrin
Lactoferrin is an iron-binding glycoprotein found in neutrophil granulocytes and is activated during acute inflammation.19 The diagnostic accuracy of fecal lactoferrin is similar to that of fecal calprotectin and is superior to CRP.29 Like fecal calprotectin, fecal lactoferrin may be influenced by the extent and location of the inflamed mucosa. However, there is limited data regarding its prognostic value.25
Novel biomarkers
Mucosal addressin cell adhesion molecule-1 (MAdCAM-1)
MAdCAM-1 is expressed by endothelial cells and stimulates intestinal inflammation by binding adhesion molecules on immune cells. Elevated MAdCAM-1 expression in tissue correlates with endoscopic and histologic evidence of inflammation. Higher levels are also noted in patients with a Mayo Endoscopic Score of one who subsequently relapse.53 These associations make it a promising biomarker for monitoring disease and stratifying relapse risk.
Vedolizumab, a biologic used to treat UC and CD, blocks the interaction of MAdCAM-1 with its integrin receptor to reduce inflammation. Vedolizumab is considered a “slow-acting” biologic due to its relatively delayed onset of action. Therefore, identifying biomarkers that could predict response to vedolizumab would be particularly helpful in avoiding long periods of ineffective treatment. MAdCAM-1, particularly its adhesion to CD4+ T cells in the peripheral blood of IBD patients, correlates with subsequent clinical response to vedolizumab therapy in small studies.54 On the other hand, if intestinal endothelial cells do not express MAdCAM-1, there will likely be no clinical response to vedolizumab.55
The OPERA study evaluated a monoclonal antibody directed against MAdCAM-1 as a potential treatment option for moderate-to-severe CD but did not achieve a greater treatment effect than placebo.56 Some suggest this finding may be related to dose effect or drug delivery methods, as vedolizumab, which utilizes a similar pathway, has proven effective in treating CD patients.57
Oncostatin M (OSM)
Oncostatin M belongs to the IL-6 cytokine family and is involved in liver repair, cardiac tissue remodeling, osteoclastogenesis, and hematopoiesis. However, excessive OSM production can contribute to skin and lung inflammation, atherosclerosis, and various cancers.58 Both OSM and its receptor, OSMR, consistently show elevated levels in both the blood and inflamed mucosa of IBD patients.9,59 A single nucleotide polymorphism on chromosome 5 in the human OSM locus is strongly associated with the risk of IBD development.58,60,61 Therefore, serum OSM testing could be a promising diagnostic biomarker for identifying IBD patients, especially those with a first-degree relative.9
Hematopoietically derived OSM appears to promote inflammatory responses by enhancing the production of chemokines, cytokines, and adhesion factors by intestinal stromal cells. Overexpression of OSM in intestinal mucosa is consistently associated with an increased risk of resistance to anti-tumor necrosis factor (TNF) therapy.58 Since up to 40% of patients do not respond to anti-TNF agents, identifying alternative therapeutic targets could reduce corticosteroid usage.60,61 Because mucosal OSM correlates closely with histopathological disease severity, it raises the question of whether OSM signal is truly predictive for lack of response to anti-TNF agents specifically or is simply a marker of a more refractory and difficult-to-treat disease. The routine use of OSM in predicting clinical response is currently limited by the fact that the mucosal signal of OSM could not be reliably translated into either whole blood or serologic OSM biomarker levels.59
In tissues with increased extracellular matrix protein deposition, such as those observed in chronic inflammation and fibrosis, OSM’s effects may be amplified due to its increased stability in such environments.58 Up to 15% of CD patients will develop fibrostenotic disease with strictures within a decade after initial diagnosis.62 Despite the widespread knowledge of this common phenomenon, there are no available therapeutic agents targeting intestinal fibrosis. Data in mouse models suggest that OSM exerts significant fibrogenic activity. However, its potential as a target for stricturing CD has not been investigated and no data have yet proven that neutralizing OSM can reverse fibrosis.60,63 Interestingly, OSM levels are also elevated in the colonic mucosa of patients with UC despite UC not being as strongly associated with fibrosis.58
OSM has been identified as a potential mediator of nociception and is associated with common comorbidities of IBD, such as psoriasis and arthritis. Thus, OSM blockage could be beneficial for IBD patients, not only in reducing gut inflammation but also in alleviating various comorbid conditions.58 Recent studies suggest that OSM may sensitize sensory afferents in IBD patients, leading to increased colonic afferent discharge. These findings suggest that OSM may contribute to the severity of abdominal pain in IBD and could be a potential target for managing chronic pain in IBD patients.64
OSM thresholds have not been established and may differ between UC and CD. This should be investigated in future studies.61 Additionally, OSM may not be predictive of disease response in pediatric patients.65 As a newly discovered biomarker, the potential value of OSM has garnered significant attention, but its potential uses and reliability in IBD require further evaluation.9
Nucleotide-binding oligomerization domain protein 2 (NOD2)
First identified in 1996 on chromosome 16, NOD2 is expressed by many leukocytes as well as Paneth cells, fibroblasts, and epithelial cells. NOD2 acts as a positive regulator of immune defense, partly by regulating autophagy.67 NOD2 is one of several susceptibility loci recognized in relation to IBD risk, but it is associated with CD risk alone.29 It has the highest expression in terminal ileal Paneth cells, supporting its role in the development of ileal disease.67 One of the crucial pathogenic mechanisms of NOD2 may involve impaired bacterial clearance. This leads to increased bacterial invasion into the mucosa, activating inflammatory pathways that contribute to the deeper, often transmural, inflammation seen in ileal CD.66,67 The intestinal microbiome may play a key role as a trigger for the inflammation seen in NOD2-related CD. Knockout NOD2 [−/−] mice did not develop spontaneous colitis in sterile conditions but only developed inflammation when introduced to bacteria.68
Interestingly, the most common NOD2 mutations occur in Caucasians, and NOD2 mutations associated with CD are not observed in Asian or sub-Saharan African populations.66 Therefore, sequencing for NOD2 variants could have important impacts for Caucasians as it could correlate with CD risk, but it is controversial for other ethnicities.29 Between 30–50% of CD patients in the Western hemisphere carry disease-causing mutations in at least one NOD2 allele. Patients with double-dose mutations typically experience disease onset at a younger age than those with no mutation.69
However, it is worth noting that normal, healthy individuals may have NOD2 mutations on both chromosomes with no evidence of active disease.69 Smoking has been proposed as a possible modulator of NOD2 mRNA expression and function, suggesting that epigenetic modification of NOD2 may confer an increased risk of developing CD through gene-environment interaction.70 NOD2 variants have been associated with a familial CD with a predisposition to stricturing disease.29 However, some studies suggest that NOD2 may not be directly associated with stricturing itself after accounting for disease location in the ileum.71 Establishing a timely diagnosis of NOD2-associated disease could allow for more targeted treatment with either new or existing therapies to prevent irreversible fibrosis or the need for surgery. However, the utility of this strategy remains hypothetical as there have been no studies to date investigating this specific use of NOD2 as a treatment decision tool.67
Anti-integrin αvβ6
Integrins are cell surface glycoprotein receptor heterodimers composed of α and β subunits. They are involved in cell signaling, proliferation, adhesion, and migration.72 Integrin αvβ6 appears to be exclusive to epithelial cells and functions to maintain the epithelial barrier. It also attenuates the innate immune system’s surveillance of the GI tract through its interaction with the extracellular matrix.36,73 Loss of epithelial barrier integrity could be an early feature of UC pathogenesis, making the appearance of anti-αvβ6 autoantibodies a potential preclinical biomarker of disease.73 While previous studies have noted reduced αvβ6 expression in the mucosa of CD patients,74 the majority of studies have focused on the correlation with UC.
Anti-αvβ6 autoantibodies were significantly higher among individuals who developed UC compared with controls up to 10 years before diagnosis in PREDICTS. The increasing prevalence of anti-αvβ6 autoantibodies is superior to that of pANCA in diagnosing and predicting disease outcomes.73 The presence of anti-integrin αvβ6 autoantibodies showed a sensitivity of 92% and a specificity of 94.8% for diagnosing UC in adult patients compared to healthy controls. Ten years before diagnosis, the anti-αvβ6 autoantibody seropositivity was 12.2%, increasing to 54% at UC diagnosis, compared to 2.7% seropositivity across multiple time points in healthy controls. Those with recently diagnosed UC and elevated anti-αvβ6 autoantibodies were at an increased risk of adverse outcomes, including hospitalization, disease extension, colectomy, systemic steroid use, and/or escalation to biologic therapy.73 These findings have been supported in multiple studies in various populations, including Japan, the United States, Sweden, and pediatric populations as well.36,73
Epigenetics
MicroRNAs (miRNAs)
MiRNAs are short, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level.75 The imbalance of miRNAs could explain the pathophysiologic processes of multiple diseases, such as arrhythmias, schizophrenia, cancer, and immune-related diseases. An ever-expanding number of serological miRNAs appear to be upregulated or downregulated in IBD.29 Deciphering this variability could serve as a non-invasive measure of disease activity. Recent studies have shown that miRNAs mediate inflammatory responses and intestinal barrier function in the pathogenesis of IBD as well as playing an important role in endoplasmic reticulum stress and interactions with gut microbiota.9,76,77
There are multiple miRNA sequences that tend to be overexpressed in patients with IBD compared to healthy controls, and some may eventually be useful in distinguishing between CD and UC for those with unspecified IBD.9,78 Comprehensive microarray profiling and quantitative PCR have been used to determine the different miRNA profiles in CD, UC, and non-IBD subjects.79 Several miRNAs that show promise in the identification and treatment of IBD include:
MiRNA-192: It appears to be downregulated in the colonic mucosa of patients with active UC. It is an important inhibitory mediator of the expression of a pro-inflammatory chemokine, macrophage inflammatory peptide 2a;79,80
MiRNA-223: It has been associated with increased inflammation of the colonic mucosa in IBD patients. It targets claudin-8, a crucial protein of the tight junctions in the intestinal mucosa, through the IL-23 pathway and impairs intestinal barrier function.81 Increased levels in circulation correlate closely with disease activity in CD and UC;
MiRNA-16: Serum expression of miRNA-16 correlates with CD localized to the small bowel as well as stenosis and penetrating forms of the disease. The activity also appears to correspond with the Crohn’s Disease Activity Index. Increased levels can also be found in extensive UC. However, miRNA-16 levels did not correlate with any treatment given in CD or UC.82
The most extensively studied miRNAs with respect to the pathogenesis of intestinal fibrosis are the miRNA-200 family, which may induce the epithelial-to-mesenchymal transition,83 and the miRNA-29 family, whose downregulation has been associated with pulmonary, cardiac, and hepatic fibrosis as well as stricturing phenotypes.84
Several research studies have shown that certain miRNAs determine the extent of glucocorticoid response in multiple diseases, including hematologic neoplasms and airway hyperresponsiveness. Further research could establish the specific roles of miRNAs in predicting glucocorticoid resistance in IBD and determine whether miRNAs could be adopted as biomarkers and/or therapeutic targets in these patients.
The utilization of miRNAs as therapeutic targets would necessitate the identification of all miRNA targets and those that are consistently dysregulated. The uptake of miRNAs beyond the target organ presents a challenging obstacle to initiating miRNA as a therapeutic intervention in diseases like IBD. Additionally, the lack of consistency between experimental processes and improper controls for normal miRNA levels present significant barriers to the utilization of miRNAs as disease biomarkers.79 Any given miRNA can regulate multiple genes, consequently, targeting a single miRNA could affect several different disease processes. Because of this, therapeutic use of miRNAs is limited due to the potential for off-target effects as well as the possibility of undesirable on-target effects.85 Many studies have investigated gut/colonic expression of miRNAs in IBD, but few have examined serum miRNAs, which will determine if they will actually be useful biomarkers in clinical practice.9 To increase the feasibility of miRNA-based therapeutics, the field needs to address miRNA-regulated genes and gene networks, efficient miRNA delivery, and develop animal models that mimic critical aspects of IBD to enable testing the physiological role of miRNA and the impact of miRNA-targeted interventions.85