Differential Effects of Anti-TNFα and Anti-α4β7 Drugs on Circulating Dendritic Cells Migratory Capacity in Inflammatory Bowel Disease

Abstract

Inflammatory bowel disease (IBD) is an idiopathic and chronic disorder that includes ulcerative colitis (UC) and Crohn's disease (CD). Both diseases show an uncontrolled intestinal immune response that generates tissue inflammation. Dendritic cells (DCs) are antigen-presenting cells that play a key role in tolerance maintenance in the gastrointestinal mucosa. Although it has been reported that DC recruitment by the intestinal mucosa is more prominent in IBD patients, the specific mechanisms governing this migration are currently unknown. In this study, the expression of several homing markers and the migratory profile of circulating DC subsets towards intestinal chemo-attractants were evaluated and the effect of biological drugs with different mechanisms of action, such as anti-TNFα or anti-integrin α4β7 (vedolizumab), on this mechanism in healthy controls (HCs) and IBD patients was also assessed. Our results revealed that type 2 conventional DCs (cDC2) express differential homing marker profiles in UC and CD patients compared to HCs. Indeed, integrin β7 was differentially modulated by vedolizumab in CD and UC. Additionally, although CCL2 displayed a chemo-attractant effect over cDC2, while biological therapies did not modulate the expression of the homing markers, we paradoxically found that anti-TNF-treated cDC2 increased their migratory capacity towards CCL2 in HCs and IBD. Our results therefore suggest a key role for cDC2 migration towards the intestinal mucosa in IBD, something that could be explored in order to develop novel diagnostic biomarkers or to unravel new immunomodulatory targets in IBD.

Keywords: anti-TNFα; biological drugs; dendritic cells; inflammatory bowel disease; intestinal mucosa; migration; vedolizumab.

Figure 1

Characterization of circulating dendritic cell subsets. (A) Dendritic cell (DC) subsets were identified by flow cytometry within singlet viable peripheral blood mononuclear cells. HLA-DR⁺CD19⁻CD14⁻ cells were further subdivided into plasmacytoid dendritic cells (pDCs) (CD123⁺ CD11c⁻) and conventional dendritic cells (cDCs) (CD11c⁺). The cDC cells were further divided based on the expressions of CD141 (cDC1) and CD1c (cDC2). (B) Phenotype of the different DC subsets from healthy controls was determined by analyzing the expressions of integrin β7, CCR2, CCR5, CCR6 and CCR9. Results are expressed as the percentage of positive cells (%) (mean ± SEM n = 13–15) referred to as a fluorescence minus one. One-way ANOVA repeated measures with the Tukey correction was applied to compare the basal expression of the different markers between pDCs, cDC1 and cDC2. p-values < 0.05 were considered significant (* < 0.05, ** < 0.01, *** < 0.001).

Figure 2

Integrin β7, CCR2, CCR5, CCR6 and CCR9 expressions on circulating dendritic cells from healthy controls and patients with inflammatory bowel disease. Dendritic cell (DC) subsets were identified as in Figure 1 in healthy controls (HCs) and in patients with active ulcerative colitis (aUC), quiescent ulcerative colitis (qUC), active Crohn’s disease (aCD) and quiescent Crohn’s disease (qCD). The expressions of β7, CCR2, CCR5, CCR6 and CCR9 were further determined in each subset within each study group. Results are shown as the percentage of positive cells (%) (mean ± SEM n = 13–15). One-way ANOVA with the Tukey correction was applied to compare integrin β7, CCR2, CCR5, CCR6 and CCR9 expression levels on pDCs, cDC2 and cDC1 between HCs and the different groups of inflammatory bowel disease patients. Furthermore, homing marker expressions were compared between quiescent and active patients for each disease in order to evaluate changes associated with the different mucosal statuses within the same disease. In addition, aUC and aCD, as well as qUC and qCD were compared in order to evaluate changes associated to different diseases with the same mucosal status. p-values < 0.05 were considered significant (* < 0.05, ** < 0.01).

Figure 3

Dendritic cell subset migration towards CCL2 is increased in patients with inflammatory bowel disease. Peripheral blood mononuclear cells from healthy controls (HCs) and patients with active ulcerative colitis (aUC), quiescent ulcerative colitis (qUC), active Crohn’s disease (aCD) and quiescent Crohn’s disease (qCD) were allowed to migrate towards gut-homing chemo-attractants, including CCL2, CCL25 and MadCam1. Numbers of migrated DC subsets (pDCs, cDC2 and cDC1), as identified in Figure 1, were further determined. All results were relativized with respect to the spontaneous migration of the cells towards a non-supplemented culture medium (basal condition, dotted line) (mean ± SEM n = 15). One-way ANOVA with the Tukey correction was applied to compare with the basal condition. p-values < 0.05 were considered significant (* < 0.05, ** < 0.01, *** < 0.001).

Figure 4

Vedolizumab downregulates the integrin β7 expression on circulating dendritic cell subsets. Dendritic cell (DC) subsets were identified as in Figure 1 and studied for the expressions of β7, CCR2, CCR5, CCR6 and CCR9 in healthy controls (HCs) and in patients with active ulcerative colitis (aUC), quiescent ulcerative colitis (qUC), active Crohn’s disease (aCD) and quiescent Crohn’s disease (qCD) following conditioning with an anti-TNF biological drug (black bars) or anti-α4β7 (grey bars). Results were compared with untreated controls (white bars). The percentages of positive cells were determined within each different subset (mean ± SEM n = 13–15). Two-way ANOVA with the subsequent post hoc correction was performed in order to compare integrin β7, CCR2, CCR5, CCR6 and CCR9 expression levels on pDCs, cDC2 and cDC1 from HCs and patients with a different disease status between cells subjected to different treatments, as described above. p-values < 0.05 were considered significant (** < 0.01).

Figure 5

Biological treatments modulate the dendritic cell subset migratory capacity. Peripheral blood mononuclear cells from healthy controls (HCs) and in patients with active ulcerative colitis (aUC), quiescent ulcerative colitis (qUC), active Crohn’s disease (aCD) and quiescent Crohn’s disease (qCD) were allowed to migrate towards gut-homing chemo-attractants including CCL2, CCL25 and MadCam1, following conditioning with anti-TNF drugs (black bars) or the anti-α4β7 drug (grey bars) and compared with non-conditioned cells (white bars). Migrated dendritic cell subsets (DC) including pDCs, cDC2 and cDC1, were identified in Figure 1. All results were relativized with respect to the migration towards a non-supplemented culture medium (basal condition, dotted line) (mean ± SEM n = 13–15). Two-way ANOVA with the subsequent post hoc comparison was performed in order to determine the migration differences within each subset and condition with respect to the basal or spontaneous migration (displayed as *) and to compare the migration within each patient group between each culture condition (displayed as #). p-values < 0.05 were considered significant (* < 0.05, ** < 0.01, *** < 0.001) (# < 0.05, ## < 0.01).