Hydroxychloroquine is associated with impaired interferon-alpha and tumor necrosis factor-alpha production by plasmacytoid dendritic cells in systemic lupus erythematosus

Abstract

Introduction: Plasmacytoid dendritic cells (pDCs) constitutively express two members of the Toll-like receptor (TLR) family, TLR-9 and TLR-7, through which they can be stimulated to produce high levels of interferon (IFN)-α, a key mediator of the pathogenesis of systemic lupus erythematosus (SLE). Given the known efficacy of hydroxychloroquine (HCQ) in the treatment of SLE, we examined its ability to inhibit such pDC function in vivo.

Methods: Peripheral blood mononuclear cells (PBMCs) from SLE subjects treated or not with HCQ and from healthy controls were stimulated with the TLR-9 agonist, CpG oligodeoxynucleotides (CpG-A ODN)-2216, and the TLR-7 agonist, imiquimod. The proportion of monocytes, B cells, myeloid dendritic cells, pDCs, and natural killer (NK) cells producing IFN-α and tumor necrosis factor alpha (TNF-α) was then analyzed by multiparameter flow cytometry.

Results: After TLR-9/7 stimulation in both SLE and healthy subjects, significant production of IFN-α and TNF-α was only observed in pDCs. TLR-7 and TLR-9 induced IFN-α and TNF-α production by pDCs from subjects with SLE was decreased relative to that found in controls (TLR-9/IFN-α, P < 0.0001; TLR-9/TNF-α P < 0.0001; TLR-7/TNF-α P = 0.01). TLR-9 and TLR-7 induced IFN-α and TNF-α production by pDCs was severely impaired in 36% (TLR-9) and 33% (TLR-7) of SLE subjects. In almost all cases, these subjects were being treated with HCQ (HCQ vs. no HCQ: impaired TLR-9/IFN-α, P = 0.0003; impaired TLR-7/IFN-α, P = 0.07; impaired TLR-9/TNF-α, P < 0.009; impaired TLR-7/TNF-α, P < 0.01).

Conclusions: Treatment with HCQ is associated with impaired ability of pDCs from subjects with SLE to produce IFN-α and TNF-α upon stimulation with TLR-9 and TLR-7 agonists.

Figure 1

Plasmacytoid dendritic cells are responsible for IFN-α and TNF-α production upon TLR-9/7 stimulation. Flow cytometric analysis of IFN-α (left) and TNF-α (right) production after TLR-9 (A) or TLR-7 (B) stimulation of monocytes (CD14+), B cells (CD20+), mDCs (CD11c+), pDCs (CD123+), and NK cells (CD16+) found in the PBMCs of SLE patients (n = 39, white circles) and healthy (n = 10, black circles) (see supplementary Figure S1 for details on phenotypic analysis). The lines represent the median IFN-α/TNF-α production by subsets of PBMCs. (C) Flow cytometric analysis of IFN-α and TNF-α production after media (No Stim, top), TLR-9 (middle) and TLR-7 (bottom) stimulation of plasmacytoid dendritic cells (pDCs), (HLA-DR+CD14-CD16-CD20-) CD123+ in one representative subject. *, refers to P < 0.05, ** refers to P < 0.005, *** refers to P < 0.0001.

Figure 2

pDCs from SLE patients show impaired production of IFN-α and TNF-α after TLR-9/7 stimulation. Comparison of the frequency of circulating pDCs (CD123⁺ cells) producing IFN-α (left) and TNF-α (right) after TLR-9 (A) or TLR-7 (B) stimulation between SLE subjects (n = 39, white circles) and healthy controls (n = 10, black circles). In each case, the lines represent the mean value and the error bars show the standard deviation.

Figure 3

Frequency of circulating pDCs, pDCs IFN-α+/TNF-α+ after TLR-9/7 stimulation and lupus disease activity. (A) Flow cytometric analysis of pDCs (HLA-DR+CD123+ cells) found in the PBMCs in one representative subject (left). Comparison of the frequency of circulating pDCs found in SLE subjects (white circles) and healthy controls (black circles) (right). (B) Absence of statistical correlation between the frequency of CD123+ pDCs IFN-α+ (white triangles) or TNF-α+ (black triangles) after TLR-9 (left) or TLR-7 (right) stimulation and disease activity assessed by using the Systemic Lupus Activity Questionnaire (SLAQ) in SLE patients (TLR-9 stimulation: IFN-α/SLAQ, r = -0.20, P = 0.23; TNF-α/SLAQ, r = -0.08, P = 0.62) (TLR-7 stimulation: IFN-α/SLAQ, r = -0.18, P = 0.28; TNF-α/SLAQ, r = -0.01, P = 0.96). In each case, the lines represent the mean value and the error bars show the standard deviation.

Figure 4

pDC production of IFN-α/TNF-α upon TLR-9/7 stimulation in SLE subjects treated with HCQ. Comparison of the frequency of pDCs (CD123⁺ cells) producing IFN-α (left) and TNF-α (right) after TLR-9 (A) or TLR-7 (B) stimulation between SLE subjects that were treated (n = 25, white squares) or not (n = 14, black triangles) with HCQ. In each case, the lines represent the mean value and the error bars show the standard deviation.

Figure 5

Monocytes and mDCs production of IFN-α/TNF-α upon TLR-4 stimulation in SLE subjects treated with HCQ. Flow cytometric analysis (left) of IFN-α and TNF-α production after TLR-4 (lipopolysaccharide, LPS) stimulation of monocytes (CD14+) (top, left) and mDCs, (CD11c+) (bottom) in a representative subject. Comparison of the frequency of monocytes (CD14+) (right top) and mDCs (CD11c+) (right bottom) cells producing TNF-α after TLR-4 stimulation between SLE subjects that were treated (white squares) or not (black triangles) with HCQ. In each case, lines represented the mean value and error bars the standard deviation.