Altered phenotype of peripheral blood dendritic cells in pediatric type 1 diabetes

Abstract

Objective: Dendritic cells (DCs) are largely responsible for the activation and fine-tuning of T-cell responses. Altered numbers of blood DCs have been reported in type 1 diabetes (T1D). We aimed at characterizing the less well-known phenotypic properties of DCs in T1D.

Research design and methods: In a case-control setting, samples from a total of 90 children were studied by flow cytometry or by quantitative real-time PCR (qPCR).

Results: We found decreased numbers of myeloid DCs (mDCs) (8.97 vs. 13.4 cells/μL, P = 0.009, n = 31) and plasmacytoid DCs (pDCs) (9.47 vs. 14.6 cells/μL, P = 0.018, n = 30) in recent-onset T1D. Using a panel of antibodies against functionally important DC markers, we detected a decreased expression of CC chemokine receptor 2 (CCR2) on mDCs (percentage above negative control, P = 0.002, n = 29) and pDCs (median intensity, P = 0.003, n = 30) from T1D patients. In an independent series of children, the reduced expression of CCR2 was confirmed by qPCR in isolated mDCs (P = 0.043, n = 20). Serum concentrations of CCR2 ligands monocyte chemotactic protein-1 and -3 did not differ between the groups. A trend for an enhanced responsiveness of the nuclear factor-κB pathway (P = 0.063, n = 39) was seen in mDCs from children with β-cell autoantibodies, which is possibly related to the reduced CCR2 expression, since CCR2 on mDCs was downregulated by nuclear factor-κB-activating agents.

Conclusions: Given the role of CCR2 in DC chemotaxis and in DC-elicited Th1 differentiation, our results may indicate a functionally important DC abnormality in T1D affecting the initiation and quality of immune responses.

Figure 1

Blood DC counts are decreased in T1D. The numbers of circulating DCs were determined by flow cytometry. The relative (A) and absolute (B) counts of mDCs were lower in recent-onset T1D patients compared with the healthy children. The relative pDC counts also tended to be lower in patients (C), and the absolute pDC counts were significantly decreased (D). ctrl, control. Horizontal lines indicate median levels.

Figure 2

CCR2 expression on blood DCs is reduced in T1D. mDCs were gated for flow-cytometric analysis as demonstrated in Supplementary Fig. 1. A representative example of CCR2 staining in patient and control (ctrl) samples is shown. Light-colored histograms indicate isotype (isot.)-control antibody levels (A). The percentage of mDCs with CCR2 expression above the isotype-control antibody-based cutoff level was decreased in T1D patients, and the MFI values for CCR2 expression were also somewhat lower in patients’ mDCs. Horizontal lines indicate median levels (B). mDCs were isolated from an independent series of recent-onset patients and control subjects for qPCR analyses. The expression of CCR2 mRNA transcripts in mDCs was decreased in patients, confirming the flow-cytometric finding. Horizontal lines indicate the means (C). CCR2 expression on pDCs, as assessed by flow cytometry, is shown in a representative patient and control sample (D). The percentage of pDCs expressing CCR2 above the isotype-control level did not differ between the groups. However, expression was significantly lower in patients, as indicated by the decreased MFI values (E). mDCs were isolated from general population blood donor buffy coats to purities >97% and stimulated with the indicated stimuli or left untreated. Overnight incubation with LPS, IL-1β, or CL097 induced a clear decrease in CCR2 expression, as measured by flow cytometry, demonstrating that CCR2 expression on human mDCs is downregulated by various DC-maturing stimuli. Individual donors are indicated with different symbols. ***P < 0.001 (F). neg, negative.

Figure 3

The responsiveness of NF-κB pathway in children with multiple diabetes-associated autoantibodies. mDCs were identified in frozen and thawed PBMC samples by multicolor flow cytometry (A). A representative example of high- and low-level NF-κB phosphorylation (pNF-κB) response to IL-1β stimulation is shown. Unstained histograms represent unstimulated samples (B). NF-κB responsiveness was more pronounced among children with β-cell autoimmunity, approaching statistical significance (C). NF-κB responses were somewhat enhanced both in recent-onset T1D patients and in autoantibody-positive children without T1D compared alone with autoantibody-negative control children, although statistical significance was not achieved (D). AAB⁺, children with multiple β-cell autoantibodies without T1D; AAB⁻, children without β-cell autoantibodies; sib, sibling; FSC, forward scatter; SSC, side scatter.