Study design and patient population
This was a prospective observational single centre explorative study in Zuyderland Medical Centre, one of the largest referral hospitals for IBD care in the Netherlands. IBD patients between 18 and 70 years old were considered eligible for this study. Per drug-classified group (outlined below), inclusion of ten patients was intended.
Exclusion criteria were patients with other auto-immune diseases, use of systemic immune suppressive drugs other than thiopurines and anti-TNF therapy for patients in remission, or proven non-adherence.
The diagnosis of IBD, comprising Crohn’s disease, ulcerative colitis and unclassified IBD (IBD-U), was based on the standard combination of clinical, biochemical, endoscopic, radiologic and histological findings. Physician global assessment (PGA) was used to define the clinical disease activity status of UC, CD and IBD-U. Patients were categorized as remission or active disease (mild, moderate or severe). All three categories of active disease were considered eligible for inclusion. In addition, PGA was objectified by biochemistry, and clinical activity scores, comprising Partial Mayo (pMayo) and Harvey-Bradshaw Index (HBI), for UC / IBD-U and CD, respectively25,26.
IBD patients were categorized into the following groups: IBD patients with active disease without IBD maintenance medication (except local or systemic corticosteroids) (1) and several drug-classified groups of IBD patients in remission on: AZA/MP monotherapy (2), TG monotherapy (3), infliximab (IFX) monotherapy (4), AZA/MP or TG and IFX combination therapy (5), and patients in remission without immune suppressive medication (6). All patients in remission with medication were on that drug regimen for at least eight weeks. Patients with active disease were included prior to initiation of thiopurine therapy.
Data from healthy subjects were obtained from the previous laboratory validation study and were used as reference values24. Healthy subjects were defined by having no known history of auto-immune diseases, no fever or other clinical complaints of active disease, no use of immune suppressive medication and a C-Reactive Protein (CRP) concentration below 10 mg/L.
Medical files of consecutive IBD patients attending the Outpatient Clinic (or the Outpatient Infusion Centre) were screened at least two weeks prior to their visit. Patients who were eligible to participate in the study were invited and blood samples for measurement of Rac1, pSTAT3 and CRP were obtained. Data on demographics, disease activity, medical records and laboratory parameters, such as baseline faecal calprotectin, cell compartments of subpopulations of leukocytes and baseline 6-TGN and 6-MMPR concentrations were collected and registered using data management (Research Manager, version 126.96.36.199). Furthermore, we objectified non-adherence of all patients based on undetectable 6-TGN and/or 6-MMPR baseline concentrations.
Peripheral ethylenediamine tetraacetic acid (EDTA) blood samples were used for immunocytochemical labelling, as recently described24. In brief, after blood collection, blood cells were washed and prepared for analysis with flowcytometry. All analyses were performed within one hour after venepuncture, since stability of Rac1 protein levels this period has been demonstrated before24. First, the cells in suspension were incubated with fluorochrome-labelled antibodies targeting extracellular antigens, followed by addition of Lyse and Fix buffer for erythrocyte lysing and fixation of the antibodies to the membrane-bound antigens. Subsequently, fluorochrome-labelled antibodies targeting intracellular antigens were added and incubated, followed by washing and resuspending cells in 1% bovine serum albumin / phosphate buffered saline (BSA/PBS) buffer for flowcytometric analysis. The samples were analysed using a BD FacsCanto flow cytometer (BD Biosciences, San Jose, USA). Lastly, a gating procedure was performed to differentiate between leukocyte subpopulations (Supplemental Data).
Rac1 and pSTAT3 protein levels were reported as fraction of positive cells as well as mean fluorescence intensity (MFI). For all IBD patients, total fractions of Rac1 and pSTAT3 protein levels were displayed as normalized MFI based on healthy subjects, reported as arbitrary units (AU). Normalized MFI was calculated by dividing an individual’s MFI by the mean MFI of healthy subjects. Conversion of raw data into AU is explained in more detail in the Supplemental Data.
Besides Rac1 and pSTAT3 protein levels, the ratio of pSTAT3 and Rac1 protein levels was calculated. This ratio was used to analyze the proportion of pSTAT3 in relation to the available Rac1. This was introduced because it was hypothesized that the inhibition of Rac1 by thiopurine therapy could in fact induce total (active and inactive) Rac1 protein expression as means of a compensation mechanism. Any potential increase of Rac1 protein expression will be corrected by the use of a ratio of pSTAT3/Rac1. Therefore, relative pSTAT3 protein levels were reported as absolute pSTAT3 protein levels divided by absolute Rac1 protein levels, correcting for absolute (inactive) Rac1 quantities.
Baseline characteristics were presented as medians with interquartile range (IQR) for non-normal distributed numerical variables and as number of patients with corresponding percentage for categorical variables. Independent Kruskal–Wallis test was used for comparison of numerical variables and Chi-square test for categorical variables. Statistical analyses were conducted using SPSS (Version 26.0). P-values of ≤ 0.05 were considered to be statistically significant.
To investigate possible differences in Rac1 and pSTAT3 protein levels between groups, statistical analyses were conducted using GraphPad Prism (version 8.0, San Diego, CA, USA). Unpaired t-tests and Mann–Whitney U tests were used for comparison of normal distributed and non-normal distributed variables, respectively. Because of the Bonferroni-correction for five independent comparisons, a corrected p-value of ≤ 0.01 (p ≤ 0.05 divided by 5) was considered statistically significant for these statistical tests.
The study was approved by the medical ethics committee of Zuyderland medical centre and Zuyd Hogeschool (METC Z, Heerlen, The Netherlands, METC-number METCZ20200110) and conforms to the ethical guidelines of the Declaration of Helsinki (2013). All participants provided written informed consent. Written informed consent was also obtained from healthy subjects.