Our investigation was focused to analyse CD14, CD16, and CD44 expression in monocyte and lymphocyte subsets, as well as granulocyte CD44 in patients with IBD treated with biological or NBT. Crohn′s disease patients treated with NBT showed decreased percentage of non-classical CD14+CD16++ monocytes, whereas patients with biological therapy remained at the control level. After biological treatment, decreased CD44 expression was detected in non-classical monocytes of ulcerative colitis patients while Crohn's disease patients monocytes were not affected. Percentage of classical CD14++CD16- monocytes was lower in the < 9 years of IBD duration subgroup compared with the longer disease duration subgroup. Patients treated with NBT of ulcerative colitis showed lower expression of CD44 in CD44+CD14+ lymphocytes, whereas for the patients treated with biological therapy there was no difference in comparison to the healthy control group. The percentage of CD44+ granulocytes was elevated in Crohn's disease compared to control subjects.
While we found no difference between biologically treated Crohn's disease and healthy controls, Nazareth et al. observed a small infliximab-dependent increase in the frequency of circulating non-classical monocytes, but without change compared to non-treated Crohn's disease patients [18]. Non-classical monocytes have the highest expression of CD16 [19]. CD16 receptor contributes to the cell activation by IgG immune complexes [20]. Consequently, non-classical monocytes are associated with adhesion, complement, and Fc gamma-mediated phagocytosis [21,22]. During IBD, the epithelial barriers of the intestines are damaged, which allows the entry of microbes into the tissue [9). Homing of non-classical monocytes via α4β7 integrin to the gut mediates macrophage-dependent intestinal wound healing [23]. These macrophages are upregulated in the proliferative phase of wound healing but decreased following vedolizumab, an anti-α4β7 integrin antibody routinely used in IBD treatment [24,25].
Therapies interfering with gut homing have an impact on monocyte subset homing. Classical monocytes preferentially home to inflamed sites in contrast to the non-classical monocytes that home to non-inflamed tissues [26]. In peripheral tissues, monocytes may develop into macrophages and monocyte-derived dendritic cells [27]. The presence of cytokines and microbial compounds can give rise to various types of macrophages: proinflammatory, which secrete cytokines like TNF-α, IL-12 or IL-23; and anti-inflammatory, which produce cytokines such as IL-10 or TGF-β [28]. The general perception is that non-classical monocytes are biased towards wound healing macrophages [23]. Decreased percentage of non-classical CD14+CD16++ monocytes in non-biologically treated Crohn's disease and their unchanged level after biological therapy indicate the higher potential of intestinal wound healing in biologically treated patients.
Biological treatment decreased CD44 expression on non-classical monocytes of ulcerative colitis patients while monocytes in patients with Crohn's disease were not affected. Total monocyte CD44 isoforms are more precisely designated as CD44s, containing variant exons (CD44v and CD44v7) in human-activated monocytes and different T-cell subpopulations [29]. In addition to hyaluronan [10-12], partner ligand molecules of CD44 receptor include laminin, hepatocyte growth factor, vascular endothelial growth factor, and osteopontin [29, 30]. Inflammation is initiated by elevated IL-6 secretion from monocytes induced after interaction between osteopontin and CD44v7 [30]. In CD44v7 knock out mice, IL-6 levels are 30 times lower compared to wild type mice, leading to reduced colonic inflammation. Expression of CD44 gene was higher in patient biopsies taken from inflamed than from non-inflamed regions [30]. CD44 binds to hyaluronan, laminin and osteopontin within the extracellular matrix after monocyte extravasation [29, 30]. Prior to the extravasation, monocyte ligand α4β7 has to bind to MAdCAM-1 receptor on endothelial cell [31]. A phase II study of fully human antibody towards MAdCAM-1 showed induction of remission and mucosal healing after 12 weeks in patients with ulcerative colitis but not with Crohn's disease, probably due to an inflammatory change that extends through the entire bowel wall [31]. We can assume the same for the unaffected CD44 expression on non-classical monocytes in Crohn's disease patients in this study.
Classical CD14++CD16- monocytes are inflammatory [32]. Elevated secretion or dysregulation of IL-6 and its signaling pathway may play a major role in the pathogenesis of IBD [33,34). With longer disease duration, inflamed colonic mucosa exhibits increasing chromosomal instability and hypermethylation, marking the colon at risk for further carcinogenesis and indicating the important role of inflammation in this setting [34, 35]. In the present study, percentage of CD14++CD16- monocytes was lower in the < 9 years of IBD duration subgroup compared with the longer disease duration subgroup, indicating better control of the disease in the first period.
In parallel to the monocyte analyses, CD14, CD16, and CD44 surface molecules were also analysed in lymphocytes [36]. Lymphocytes exhibit low levels of CD14 staining. Among 21 different lymphocyte subpopulations, Kalina et al. detected CD14 only at B naive tonsil cells [37]. Naive B cells continuously recirculate throughout the body [38]. Upon extravasation from the peripheral circulation through endothelial vesicles found in the lymphoepithelium into the secondary lymphoid tissue, such as the tonsils, B cells remain there for a few days and then re-enter the circulation unless they encounter cognate antigen [22]. In this study, we detected higher CD44 expression in minor CD44+CD14+ lymphocyte subpopulation in biologically treated ulcerative colitis compared to NBT. NBT resulted in lower CD44 expression compared to the control group, while the biologically treated and control group had similar CD44 expression. In ulcerative colitis, blood lymphocytes include circulating naive and memory T and B lymphocytes, as well as primed effector cells that are en route to the inflamed gut mucosa [39]. Naive cells constitute more than half of the blood B cells in healthy adults. Patients with ulcerative colitis and those with Crohn's disease show increased percentages of CD23+ B cells, which are recognized as being naive B cells [39]. However, in ulcerative colitis, gut inflammation and activation of T cells appear to be a hallmark of the disease. In Crohn's disease, arrested B cell activation beyond the naive stage seems to be a characteristic sign [39]. Colonic mucosa samples from patients with ulcerative colitis lack naive B cells that are present in samples from patients with active Crohn's disease [39]. We can speculate that lower CD44 expression in CD44+CD14+ lymphocyte in blood, after NBT, found in ulcerative colitis but not in Crohn's disease, can be due to the higher naive B cell count in ulcerative colitis. Excessive production of IL-1, IL-6, IL12/23, and TNFα mediate distinct abnormal T-cell responses resulting in tissue fibrosis [40].
The expression of CD44 per one granulocyte was unaffected in both IBD groups of our study, but the percentage of CD44+ granulocytes was elevated in Crohn's disease compared to control and specifically in biologically treated patients with Crohn's disease. Lampinen et al. described a significantly larger percentage of CD44high eosinophils in patients with active Crohn's disease and ulcerative colitis compared with control subjects [41]. CD44high eosinophils from collagenous colitis intestinal biopsy samples have higher CD66b expression in activated state. Budesonide treatment restores the normal activation of eosinophils [42]). The role of neutrophils in the gut differs between Crohn's disease and ulcerative colitis [23]. In ulcerative colitis, unrestricted neutrophil activation cause tissue damage, whereas in Crohn's disease, defective neutrophils are not able to limit invasion by microorganisms, leading to uncontrolled inflammatory reaction [43-45].
Biological therapy lowered erythrocyte sedimentation rate (ESR) and fecal calprotectin in ulcerative colitis in comparison to control, while these parameters were unchanged in patients with Crohn's disease. Furthermore, elevated fecal calprotectin was positively associated whit inflammatory biomarkers, ESR and CRP [46]. Higher fecal calprotectin and the presence of mucus in diarrhea are known as significant predictors of the development of Crohn's disease and disease activity [47, 48]. In the multivariate analyses, only monocyte count and fecal calprotectin were associated with relapse [49]. NBT lowered white blood cells in patients with ulcerative colitis in our study rendering them more vulnerable to infections.
Biological therapy represents a significant improvement in the treatment of IBD as it can inhibit the inflammatory cascade in chronic inflammatory disease, and thus change the course and stop the progression of the disease. However, the possible side effects are numerous and can affect almost any organ system, some of which can be very serious, such as the development of serious infections, pulmonary embolism of systemic and respiratory hypersensitivity, gastrointestinal fistulas, and malignant and lymphoproliferative diseases.
Understanding the fundamental differentiation and regulation of monocyte and lymphocyte cells will dictate future therapeutic effects, reducing their effect when they are harmful and amplifying their effect when they are useful. This study has several limitations. The design of the present study was cross-sectional, which impedes the establishment of a true causal relationship. Second, this study had a relatively small number of participants. The IBD group from our study mostly was heterogenous in disease activity which additionally decreases the sample size by stratification and impedes the generalization of the findings to the entire IBD population. IBD is a repeating disease and the pattern of excreted markers is not constant, so one measurement is not enough to determine a causal relationship. In the preparation of the study, we used only the parameter hs-CRP, other inflammatory parameters that play an important role in the results in the preparation of study were not measured.