Functional impairment of human myeloid dendritic cells during Schistosoma haematobium infection

Abstract

Chronic Schistosoma infection is often characterized by a state of T cell hyporesponsiveness of the host. Suppression of dendritic cell (DC) function could be one of the mechanisms underlying this phenomenon, since Schistosoma antigens are potent modulators of dendritic cell function in vitro. Yet, it remains to be established whether DC function is modulated during chronic human Schistosoma infection in vivo. To address this question, the effect of Schistosoma haematobium infection on the function of human blood DC was evaluated. We found that plasmacytoid (pDC) and myeloid DC (mDC) from infected subjects were present at lower frequencies in peripheral blood and that mDC displayed lower expression levels of HLA-DR compared to those from uninfected individuals. Furthermore, mDC from infected subjects, but not pDC, were found to have a reduced capacity to respond to TLR ligands, as determined by MAPK signaling, cytokine production and expression of maturation markers. Moreover, the T cell activating capacity of TLR-matured mDC from infected subjects was lower, likely as a result of reduced HLA-DR expression. Collectively these data show that S. haematobium infection is associated with functional impairment of human DC function in vivo and provide new insights into the underlying mechanisms of T cell hyporesponsiveness during chronic schistosomiasis.

Figure 1. Reduced frequencies of mDC and pDC in blood from S. haematobium-infected subjects.

(A) Blood DC were identified in fixed PBMC as HLA-DR⁺/CD14⁻/CD19⁻ cells and subsequently subdivided into mDC and pDC on the basis of positive staining for BDCA-1 and BDCA-2, respectively. Data from one representative donor is shown. (B) Frequencies of blood DC subsets in total PBMC (C) and their surface expression of HLADR, CD80, CD86 and CCR7 was determined by following the gating strategy shown in (A). (B) Box and whiskers with 10–90% percentile are shown. (C) Bars represent mean + SD. (B+C) Each group represents data from 20 donors.

Figure 2. mDC, but not pDC, from S. haematobium-infected subjects have impaired TLR responses.

Isolated blood DC were stimulated with 100 ng/ml LPS (mDC), 1 µg/ml R848 (mDC & pDC) or 1 µg/ml CpG (pDC) for 40 h. (A–D) LPS- and R848-matured mDC and CpG and R848 stimulated pDC were analysed for maturation marker expression by FACS. Expression of surface marker is either shown (A+C) as fold increase relative to medium or (B+D) as absolute mean fluorescence intensity. (E) Cytokines levels in 40 h culture supernatants from TLR-stimulated DC were determined by multiplex Luminex. Values represent cytokines concentration from which medium control cytokines levels have been subtracted. (A+C) box plots represent 25–75 percentile range with error bars showing minimum to maximum. *, p<0,05; **, p<0,01; ***, p<0,001 for significant differences in fold increase in maturation marker expression relative to the medium-stimulated DC. (B, D and E) Bars represent mean + SD. Each group represents data from 20 donors, except for (E) in which R848-stimulated pDC cytokine data are represented by 4 and 5 donors for the infected and un-infected group, respectively. ND: not detectable.

Figure 3. mDC from S. haematobium-infected subjects have an altered MAPK signaling, but not TLR4 expression profile.

(A) TLR4 expression was analysed on mDC present in PBMC following the gating strategy as described in legend of Fig. 1. (B and C) 20 and 60 minutes after stimulation with LPS, mDC were intracellularly stained for (B) phospophorylated p38 and ERK and (C) the ratio between p-ERK and p-p38 was determined by dividing the respective mean fluorescence intensities per sample. Each group represents data from 5 donors. (A) Bars represent mean + SD. (B and C) box plots represent 25–75 percentile range with error bars showing minimum to maximum.

Figure 4. mDC from S. haematobium-infected subjects have a reduced T cell activating capacity due to lower HLA-DR expression.

LPS matured mDC were cocultured with allogeneic naïve T helper cells for 6 d after which (A) T cell expansion was determined with a counting chamber and (B) Intracellular cytokine production or (C) CD25 and HLA-DR expression was assayed by FACS, 6 h after restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A for the last 2 h. (D+E) LPS-matured mDC from European controls were cocultured with allogeneic naïve T helper cells for 6 d in the presence of depicted neutralizing antibodies and T cells were counted as in (A). T cell counts are shown as (D) absolute values or (E) relative to the control condition. (A,B,D) Horizontal bars represent mean based on data from 9 donors in each group. (C) Box plots represent 25–75 percentile range with error bars showing minimum to maximum based data of 9 donors in each group. (E) Bars represent mean + SD of 3 independent experiments.