Patients with active inflammatory bowel disease lack immature peripheral blood plasmacytoid and myeloid dendritic cells

Abstract

Background: Breakdown of tolerance against the commensal microflora is believed to be a major factor in the pathogenesis of inflammatory bowel disease (IBD). Dendritic cells (DC) have been implicated in this process in various animal models, but data on human DC in IBD are very limited.

Aim: To characterise plasmacytoid DC (PDC) and myeloid DC (MDC) in patients with active versus inactive IBD and healthy controls.

Patients and methods: Peripheral blood was obtained from 106 patients (Crohn's disease (CD) n=49, ulcerative colitis (UC) n=57) and healthy controls (n=19). Disease activity was scored using the modified Truelove Witts (MTWSI) for UC and the Harvey Bradshaw severity indices (HBSI) for CD. Four colour flow cytometric analysis was used to identify, enumerate, and phenotype DC. DC from patients with acute flare ups and healthy controls were cultured and stimulated with CpG ODN 2006 or lipopolysaccharide (LPS).

Results: IBD patients in remission (PDC UC, 0.39%; CD, 0.35%; MDC-1 UC, 0.23%; CD, 0.22% of PBMC) have slightly lower numbers of circulating DC compared with healthy controls (PDC 0.41%, MDC-1 0.25% of PBMC). In acute flare ups IBD patients experience a significant drop of DC (PDC UC, 0.04%; CD, 0.11%; MDC-1 UC, 0.11%; CD, 0.14% of PBMC) that correlates with disease activity (correlation coefficients: PDC MTWSI, 0.93; HBSI, 0.79; MDC-1 MTWSI, 0.75; HBSI, 0.81). Moreover, both express alpha4beta7 integrin and display an immature phenotype. Freshly isolated PDC and MDC-1 from untreated flaring IBD patients express higher baseline levels of CD86 which increases further in culture and upon stimulation compared with healthy controls.

Conclusion: IBD patients lack immature blood DC during flare ups which possibly migrate to the gut. An aberrant response to microbial surrogate stimuli suggests a disturbed interaction with commensals.

Figure 1

Inflammatory bowel disease medications summary. Bars show percentages.

Figure 2

FACS plots from representative experiments illustrating the significant drop of peripheral blood dendritic plasmacytoid (PDC  =  PI-(vital), CD123+, BDCA-2+ cells) and myeloid dendritic (MDC-1  =  PI-(vital), CD14-, CD19-, CD11c+, BDCA-1) cells in patients with active ulcerative colitis and Crohn’s disease. The same trend was seen with MDC-2 (data not shown). Ulcerative colitis PDC: (A) remission v (B) flare up: 0.66% PDC v 0.06% PDC). Ulcerative colitis MDC-1: (C) remission v (D) flare up: 0.25% MDC-1 v 0.06% MDC-1. Crohn’s disease PDC: (E) remission v (F) flare up: 0.31% PDC v 0.12% PDC. Crohn’s disease MDC-1: (G) remission v (H) flare up: 0.23% MDC-1 v 0.06% MDC-1.

Figure 3

Assessment of circulating plasmacytoid dendritic cells (PDC) in IBD. (A) IBD patients in remission have slightly lower numbers of circulating peripheral blood plasmacytoid dendritic cells compared with healthy controls. In IBD flare-ups PDC drop significantly. Data is expressed as mean percentage of vital PBMC. (B) The fraction of circulating PDC correlates (CC: 0.93) with the modified Truelove Witts severity index (MTWSI) in patients with ulcerative colitis. (C) The fraction of circulating PDC shows a high correlation (CC: 0.80) with the Harvey Bradshaw severity index (HBSI) in patients with Crohn’s disease.

Figure 4

Assessment of circulating myeloid dendritic cells (MDC) in IBD. (A) IBD patients in remission have slightly lower numbers of circulating peripheral blood myeloid (MDC-1) dendritic cells compared with healthy controls. In IBD flare ups MDC drop significantly. Data are expressed as mean percentage of vital PBMC. (B) The fraction of circulating MDC correlates (CC: 0.75) with the modified Truelove Witts severity index (MTWSI) in patients with ulcerative colitis. (C) The fraction of circulating MDC shows a high correlation (CC: 0.81) with the Harvey Bradshaw severity index (HBSI) in patients with Crohn’s disease.

Figure 5

FACS analysis of maturation markers on PDC and MDC in heparinised whole blood. (Stars and asterisks denote extremes and outliers, respectively.) (A) PDC and (B) MDC-1 display an immature phenotype that does not change in acute flare ups.

Figure 6

PDC, MDC-1, and MDC-2 express the gut homing marker α4β7 integrin. Example of a patient with Crohn’s disease (expression in ulcerative colitis is similar (data not shown)). (A) 93.6% of all vital MDC-1 are double positive for α4β7 integrin. (B) 67.1% of all vital PDCs are double positive for α4β7 integrin. (C) 98.2% of all vital MDC-2 are double positive for α4β7 integrin.

Figure 7

Histograms summarising the regulation of the co-stimulatory molecule CD86, a DC maturation marker, in healthy controls (dotted line), and IBD patient PDC and MDC-1, freshly isolated, 24 hour cultured, or stimulated (dotted line) as well as isotype matched controls (bold line). Plots are representative for five independent experiments. Cultured PDC and MDC-1 from untreated IBD patients with acute flare ups express higher levels of the co-stimulatory molecule CD86 compared with healthy controls. CD86 is upregulated faster and to a higher level upon stimulation with microbial surrogate stimuli such as CpG ODN 2006 or LPS, respectively.