Introduction
In the evaluation of the effectiveness of anti-inflammatory therapy in Crohn’s disease (CD) in recent years, mucosal healing (MH) has emerged as an increasingly important therapeutic goal in reducing clinical relapse rates, hospitalization rates, and surgical need [1]. Mucosal healing, a concept introduced especially after the emergence of biologicals, is an important endpoint in sustained clinical remission of CD, and therefore might be accepted as a goal for CD treatment [2–4].
Histones H3 and H4 are nuclear proteins with antimicrobial activities and are considered as a component of the innate immune system [5]. Serum levels of histones are significantly elevated and possibly correlate with the severity or poor prognosis of several disorders such as acute bacterial infection, sepsis, autoimmune diseases (e.g systemic lupus erythematosus), cerebral stroke, trauma, and cancer. It has been described that histone H4 can activate Toll-like receptors 2 and 4 resulting in downstream cytokine production, leukocyte recruitment, microvascular leakage, inflammation, and tissue injury in murine models [6–8]. Therefore, extracellular histones are thought to be used as biomarkers [9]. It is stated that the level of circulating histones can be a novel biomarker in predicting disease severity and mortality in acute liver failure and in acute pancreatitis [10, 11]. However, the severity of pancreatitis was not found to be related with serum H3 and H4 [12].
Currently, a noninvasive biomarker of MH in CD is needed. In previous studies, histones have been reported to increase in inflammatory bowel diseases. Tsaprouni et al. studied acetylated histone H4 levels in animal models and human tissues taken by ileocolonoscopic biopsy in patients with CD. They found that histone H4 acetylation on CD was associated with inflammation, and they stated that histone H4 may constitute a therapeutic target for mucosal inflammation [13].
Aim
Thus, we aimed to investigate the role of serum histone H4 in distinguishing between those with and those without MH in ileocolonic CD patients.
Material and methods
Patients
Our study was an observational, preliminary study and was conducted in the Department of Gastroenterology at Bezmialem Vakıf University Faculty of Medicine between January 2017 and February 2018.
A total of 44 patients with a diagnosis of ileocolonic CD, who were followed up at the IBH outpatient clinic of our hospital and who applied to the endoscopy unit for ileocolonoscopic evaluation, were included in the study. Exclusion criteria included infectious or other inflammatory diseases, and heart, liver, and kidney failure. In addition, 26 age- and sex-matched control patients who were admitted to the outpatient clinic with nonspecific complaints but no prominent pathology were enrolled. The study protocol was approved by the Bezmialem Vakif University local Ethics Committee (71306642-050.01.04). Written informed consent was obtained from the enrolled patients.
Peripheral blood samples (3 ml) were taken from the ileocolonic CD patients and from the healthy control group. The blood samples were centrifuged in sodium citrate tubes at 3000 rpm for 20 min. Then the separated serums were kept at –80°C until analysis time. Clinical features, demographic data, biochemistry, and haemogram results of the patients were recorded from the electronic database.
Blood assay
Histone H4 was measured with a sandwich enzyme-linked immunosorbent assay (ELISA) using commercial kits (lot no: 71135C-H1658C094; MyBioSource, Inc., San Diego, CA, USA) and an ELISA microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The assay was performed in 96-well plates pre-coated with anti-human histone H4 acetylated on lysine 16 (HIST1H4) antibodies according to the manufacturer’s recommendations. Biotin-conjugated HIST1H4A antibodies were used as detection antibodies. Standards, test samples, and biotin conjugated detection antibodies were added to the wells subsequently and washed with wash buffer. Horseradish peroxidase (HRP)-Streptavidin was then added, and unbound conjugates were washed away with wash buffer. 3,3’,5,5’-Tetramethylbenzidine (TMB) was used to visualize the HRP enzymatic reaction. In the presence of HRP, TMB generates a blue product, converted into a yellow compound after the addition of an acidic stop solution. The intensity of the yellow was proportional to the amount of HIST1H4A in the plate. The absorbance was read at 450 nm in a microplate reader, and the concentration of HIST1H4A in the samples was calculated using a standard curve. The results were expressed in ng/ml.
Mucosal healing
Mucosal healing is simply defined as the absence of ulceration and erosions [14, 15]. Also, the largest trials that used MH as the primary or secondary endpoint, have used the definition of absence of ulcers instead of the prespecified CDEIS or SES-CD cut-off values [14]. In this study, as reported by Baert et al., we used the definition of MH as the absence of mucosal erosions or ulcers (a SES-CD score of 0), and we defined the absence of MH as the presence of any size of superficial or deep ulcers or erosions on the mucosal surface (a SES-CD score of ≥ 1) [16].
In addition, patients were questioned about abdominal cramping pain, diarrhoea, and weight loss, which are among the characteristic features of CD before ileocolonoscopic evaluation [17]. Accordingly, those with the above complaints were recorded as “symptomatic” and those without were recorded as “asymptomatic”.
Statistical analysis
IBM SPSS Statistics 25.0 package 25.0 (IBM Corp., Armonk, NY, USA) was used. The Kolmogorov-Smirnov test was used to assess the normal distribution of the parameters. Quantitative variables were calculated as mean (interval) or median (interquartile range), and the qualitative variables were calculated as a percentage. Comparison of the quantitative variables with a nonparametric distribution was performed by the Mann-Whitney test. The Mann-Whitney test was used to compare the averages since the distribution was compatible with nonparametric variables other than FC. Comparison of qualitative data was done with the 2 test. Spearman’s rho correlation analysis was used to examine the relationship between parameters with non-normal distribution. Values below p < 0.05 were considered statistically significant.
Results
Forty-four consecutive patients with ileocolonic CD and 26 age- and gender-matched healthy control group’s demographic details are shown in Table I.
Endoscopic activity assessment
We compared serum histone H4 levels as well as available recorded surveillance markers as found in the recording system such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) between groups with and without MH in patients with ileocolonic CD (Table II).
In Table III, we compared CRP, ESR, and histone H4 levels between patients with abdominal pain or bowel movement disorder and those who were symptom free.
Serum histone H4 levels were significantly different between those with and without MH, and between those with and without symptoms. However, when we compared those with MH with respect to the presence of symptoms, the p-value was close to the limit of significance, but not significant (p = 0.051) (Table IV).
According to our data MH cannot be expected in all asymptomatic patients. Therefore, it can be thought that some of the patients who do not have MH continue to have the disease without symptoms.
Serum histone H4 levels according to CD behaviours, MH, and symptoms
Comparison of serum histone H4 levels between patients diagnosed with ileocolonic CD and the healthy control group are shown in Table V. Also, the comparisons between CD behaviour, MH, and symptoms are shown in Table V.
Serum Histone H4 levels were significantly higher in patients diagnosed with ileocolonic CD compared to the control group (p = 0.002). Also, serum histone H4 levels were significantly lower in asymptomatic patients (p = 0.033) and in patients with MH (p = 0.028).
Serum histone H4 levels do not correlate with CRP and ESR
We did not find a significant correlation between serum histone H4 and CRP (r = 0.118, p = 0.456) or ESR (r = 0.113, p = 0.480).
Discussion
The most desired treatment endpoint is the resolution of mucosal inflammation in CD. One of the scores used to evaluate CD activity is the Crohn’s Disease Activity Index (CDAI), which is considered the gold standard for clinical trials [18]. But this method was not found to be a reliable measure of the underlying inflammation [19]. Two different models are used in the severity of the endoscopic lesion scoring. The first of these is Crohn’s Disease Endoscopic Index of Severity (CDEIS), which is mostly used in randomised studies [20]. The second method is the Simple Endoscopic Score of Crohn’s Disease (SES-CD), which is easier to apply and more useful in routine clinical practice [21]. With the introduction of biological agents, achieving MH has become a treatment goal by decreasing the need for surgery and reducing hospitalization rates in CD [22].
According to an expert consensus report published in 2012, it is stated that MH must be regularly monitored, and endoscopy is a gold standard for assessing MH accurately [1]. These recommendations are still valid. Endoscopic assessment with objective evaluation of MH has become an essential part in tailoring the treatment [15]. Therefore, decisions regarding medical therapy can be guided by the techniques or markers showing MH, which is still under investigation. Biomarkers such as FC have yet to demonstrate sufficient specificity to replace endoscopic evaluation for mucosal healing [23]. Also, CRP and ESR are not sufficiently specific to monitor CD activity; only endoscopic assessment was stated to be effective [24].
In this study, according to the results of ileocolonoscopic evaluation, we divided the patients into 2 groups: those who were considered to have MH and those who were not considered to have MH. First, we compared serum histone H4, CRP, and ESR levels between those with and those without MH. Accordingly, we found that serum histone H4 levels were significantly lower in patients with MH (p = 0.028) while CRP and ESR values did not differ (p = 0.281, and p = 0.203, respectively) between the groups. In ileocolonic CD patients, CRP and serum histone H4 levels were significantly higher compared to the control group (p = 0.024 and p = 0.002, respectively). Also, in terms of the presence of symptoms, there was no difference between CRP and ESR among the asymptomatic and symptomatic patients in CD patients (0.779 and 0.652, respectively), but serum histone H4 levels were significantly higher in those who were symptomatic (p = 0.033). Finally, we compared those with MH according to the presence of symptoms; however, we could not find a significant relationship between MH and the presence of disease symptoms (p = 0.051). According to this comparison result, a lack of symptoms does not mean that MH is present.
Therefore, as a new biomarker candidate serum, histone H4 differed significantly between those with and without MH, and those with and without symptoms in ileocolonic CD (Table IV). To the best of our knowledge, our report is the first to investigate the prediction of MH in CD patients by using serum histone H4 as a biomarker.
In earlier studies, dexamethasone was reported to suppress histone H4 acetylation induced by TNF- [25]. Also, in previous studies, Tsaprouni et al. reported that serum histone H4 significantly increased in the disease involvement areas in animal models and CD patient biopsies, but not in areas without disease [13]. Our results are in agreement with this experimental model data. We observed that serum histone H4 levels were found to be significantly lower in patients who were found to have MH endoscopically compared to the group found to have no MH.
The limitations of the study are first that our study was a single-centre, preliminary study. Second, ethics committee approval was given only for the histone kit for this study, and no budget could be allocated for other blood tests. Therefore, other examinations related to the groups could be recorded retrospectively, as found in the recording system. Accordingly, CRP was available in 42 patients, and ESR was available in 40 patients. However, since the main purpose of the study was to evaluate the relationship of MH with histone H4 levels, we think that the absence of CRP and ESR results in some patients will not affect the serum histone H4 –MH assessment. The third limitation of the study was that the histone H4 kits were available as an experimental kit, so cut-off values could not be calculated for MH prediction.
Conclusions
Serum histone H4 may serve as a novel biomarker for predicting MH and may reduce the need for endoscopy in a patient evaluated for MH. Standardized validation studies involving a large number of patients are required.
Conflict of interest
The authors declare no conflict of interest.
References
1. Feagan BG, Lemann M, Befrits R, et al. Recommendations for the treatment of Chron’s disease with tumor necrosis factor antagonists: an expert consensus report. Inflamm Bowel Dis 2012; 18: 152-60.
2.
Mazzuoli S, Guglielmi FW, Antonelli E, et al. Definition and evaluation of mucosal healing in clinical practice. Dig Liv Dis 2013; 45; 969-77.
3.
Rutgeerts P, Vermeire S, Van Assche F. Mucosal healing in inflammatory bowel disease: impossible ideal or therapeutic target? Gut 2007; 56: 453-5.
4.
Pineton de Chambrun G, Peyrin-Biruoulet L, Lemann M, et al. Clinical implications of mucosal healing for the management of IBD. Nat Rev Gastroenterol Hepatol 2010; 7: 15-29.
5.
Tagai C, Morita S, Shiraishi T, et al. Antimicrobial properties of arginine- and lysine-rich histones and involvement of bacterial outer membrane protease T in their differential mode of actions. Peptides 2011; 32: 2003-9.
6.
Xu J, Zhang X, Monestier M, et al. Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury. J Immunol 2011; 187: 2626-31.
7.
Allam R, Scherbaum C, Darisipudi M, et al. Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4. J Am Soc Nephrol 2012; 23: 1375-88.
8.
Semeraro F, Ammollo C, Morrissey J, et al. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4. Blood 2011; 118: 1952-61.
9.
Chen R, Kang R, Fan XG, et al. Release and activity of histone in diseases. Cell Death Disease 2014; 5: e1370.
10.
Wen Z, Lei Z, YAo L, et al. Curculating histones are major mediators of systemic inflammation and cellular injury in patients with acute liver failure. Cell Death Disease 2016; 7: e2391.
11.
Szatmary P, Liu T, Abrams ST, et al. Systemic histone release distrups plasmalemma and contributes to necrosis in acute pancreatitis. Pancreatology 2017; 17: 884-92.
12.
Biberci Keskin E, İnce AT, Gültepe BS, et al. The relationship between serum histone levels and the severity of acute pancreatitis. Turk J Gastroenterol 2019; 30: 807-10.
13.
Tsaprouni LG, Ito K, Powell JJ, et al. Differential patterns of histone acetylation in inflammatory bowel diseases. J Inflammation 2011; 8: 1.
14.
Walsh A, Palmer R, Travis S. Mucosal healing as a target of therapy for colonic inflammatory bowel disease and methods to score disease activity. Gastrointest Endosc Clin N Am 2014; 24: 367-78.
15.
Klenske E, Bojarski C, Waldner M, et al. Targeting mucosal healing in Crohn’s disease: what the clinician needs to know. Ther Adv Gastroenterol 2019; 12: 1756284819856865.
16.
Baert F, Moortgat L, Van Assche G, et al. Mucosal healing predicts sustained clinical remission in patients with early-stage Crohn’s disease. Gastroenterology 2010; 138: 463-8.
17.
Khanna R, Nelson SA, Feagan BG, et al. Endoscopic scoring indices for evaluation of disease activity in Crohn’s disease. Cochrane Database Syst Rev 2016; 8: CD010642.
18.
Thia K, Faubion WA, Loftus EV, et al. Short CDAI: development and validation of a shortened and simplified Crohn’s disease activity index. Inflamm Bowel Dis 2011; 17: 105-11.
19.
Peyrin-Biroulet L, Reinisch W, Colombel JF, et al. Clinical disease activity, C-reactive protein normalisation and mucosal healing in Crohn’s disease in the SONIC trial. Gut 2014; 63: 88-95.
20.
Mary JY, Modigliani R. Development and validation of an endoscopic index of the severity for Crohn’s disease: a prospective multicentre study. Groupe d’Etudes Therapeutiques des Affections Inflammatoires du Tube Digestif (GETAID). Gut 1989; 30: 983-9.
21.
Daperno M, D’Haens G, Van Assche G, et al. Development and validation of a new, simplified endoscopic activity score for Crohn’s disease: the SES-CD. Gastrointest Endosc 2004; 60: 505-12.
22.
Peyrin-Biroulet L, Ferrante M, Magro F, et al.; Scientific Committee of the European Crohn’s and Colitis Organization. Results from the 2nd Scientific Workshop of the ECCO. I: Impact of mucosal healing on the course of inflammatory bowel disease. J Crohns Colitis 2011; 5: 477-83.
23.
Walsh A, Palmer R, Travis S. Mucosal healing as a target of therapy for colonic inflammatory bowel disease and methods to score disease activity. Gastrointest Endoscopy Clin N Am 2014; 24: 367-78.
24.
Sandborn WJ, Hanauer S, Van Assche G, et al. Treating beyond symptoms with a view to improving patient outcomes in inflammatory bowel diseases. J Crohn’s Colitis 2014; 8: 927-35.
25.
Matthews JG, Ito K, Barnes PJ, et al. Defective glucocorticoid receptor nuclear translocation and altered histone acetylation patterns in glucocorticoid-resistant patients. J Allergy Clin Immunol 2004; 113: 1100-8.
