Increased Myeloid Dendritic Cells and TNF-α Expression Predicts Poor Response to Hydroxychloroquine in Cutaneous Lupus Erythematosus

Abstract

Although antimalarials are the primary treatment for cutaneous lupus erythematosus, not all patients are equally responsive. We investigated whether different inflammatory cell population and cytokine profiles in lesional cutaneous lupus erythematosus skin could affect antimalarial responsiveness, and whether hydroxychloroquine (HCQ) and quinacrine (QC) differentially suppress inflammatory cytokines. Cutaneous lupus erythematosus patients were grouped according to their response to antimalarials (HCQ vs. HCQ+QC). On immunohistochemistry, only the myeloid dendritic cell population was significantly increased in the HCQ+QC group compared to HCQ group. While the IFN scores calculated for the selected type I IFN-regulated genes (LYE6, OAS1, OASL, ISG15, and MX1) were significantly higher in the HCQ group than the HCQ+QC group, the TNF-α level was higher in the HCQ+QC group. QC was more effective than HCQ at inhibiting the toll receptor-mediated production of TNF-α and IL-6 in the peripheral blood mononuclear cells isolated from cutaneous lupus erythematosus patients, whereas QC and HCQ inhibited IFN-α equally. QC also suppressed phospho-NF-κB p65 more profoundly than HCQ. In conclusion, increased myeloid dendritic cell population with higher TNF-α expression might contribute to HCQ refractoriness and a better response to QC. Differential suppressive effects of HCQ and QC could also affect antimalarial responses in cutaneous lupus erythematosus patients.

Figure 1.. Inflammatory cell population in lesional lupus skin varies with the treatment response.

In immunohistochemical analysis, (a) CD11c, (b) CD123, (c) MPO, and (d) MAC387 were stained to evaluate myeloid dendritic cells, plasmacytoid dendritic cells, neutrophils, and macrophages, respectively. Left two columns are representative photomicrographs of immunohistochemical staining of CD11c, CD123, MPO, and MAC387 in hydroxychloroquine (HCQ, n = 16) and hydroxychloroquine + quinacrine (HCQ+QC, n = 23) groups. Scale bar = 50 μm. HPF, high-power field; MPO, myeloperoxidase. Graphs show mean ± SEM. **P < 0.01.

Figure 2.. Inflammatory cytokine gene expression in lesional lupus skin.

Gene expression of type I IFN signatures (LYE, OAS1, OASL, ISG15, and MX1), type II IFN signatures (IL18BP), and other inflammatory cytokines important in lupus pathogenesis (TNF and IL1B) were evaluated by real-time PCR. The mRNA of type I IFN signatures were significantly upregulated in the hydroxychloroquine (HCQ) group (n = 10) compared to the hydroxychloroquine + quinacrine (HCQ+QC) group (n = 11) (LYE P = 0.0037; OAS1 P = 0.0002; OASL P = 0.0067; ISG15 P = 0.01; and MX1 P = 0.0083), with a significantly higher IFN score calculated for 5 type I IFN–regulated genes in the HCQ group, while TNF was significantly higher in the HCQ+QC group (P = 0.003). The graphs show mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.. Effects of HCQ and QC on intracellular TLR-mediated inflammatory cytokine production.

Peripheral blood mononuclear cells isolated from five cutaneous lupus erythematosus patients were stimulated with (a–c) dsRNA, (d–f) IMQ, and (g–i) ssRNA to evaluate the TLR3-, TLR7-, and TLR8-mediated production of (a, d, g) TNF-α, (b, e, h) IL-6, and (c, f, i) IFN-α by ELISA. The graphs show mean ± SEM and natural log of raw ELISA concentration values of each cytokine (n = 5). dsRNA, double-stranded RNA; HCQ, hydroxychloroquine; IMQ, imiquimod; QC, quinacrine; ssRNA, single-stranded RNA; TLR, toll-like receptor. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4.. Effects of HCQ and QC on NF-κB activation.

Peripheral blood mononuclear cells isolated from five cutaneous lupus patients were treated with increasing concentrations of QC (0.25–1 μg/ml) and HCQ (1 μg/ml) for 3 hours, with or without ssRNA stimulation. (a) TNF-α level in the culture medium after 3 hours was evaluated by ELISA. (b) Representative band from Western blot analysis for activated NF-κB (phospho-p65). GAPDH was used as the loading control. (c) The density of each band was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD) and presented as fold changes normalized to GAPDH. p-p65, phospho-p65. The graphs show mean ± SEM (n = 5). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCQ, hydroxychloroquine; QC, quinacrine; ssRNA, single-stranded RNA. ***P < 0.001.