Cytokine production by activated plasmacytoid dendritic cells and natural killer cells is suppressed by an IRAK4 inhibitor

Abstract

Background: In systemic lupus erythematosus (SLE), immune complexes (ICs) containing self-derived nucleic acids trigger the synthesis of proinflammatory cytokines by immune cells. We asked how an interleukin (IL)-1 receptor-associated kinase 4 small molecule inhibitor (IRAK4i) affects RNA-IC-induced cytokine production compared with hydroxychloroquine (HCQ).

Methods: Plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. PBMCs from SLE patients and healthy individuals were depleted of monocytes. Cells were stimulated with RNA-containing IC (RNA-IC) in the presence or absence of IRAK4i I92 or HCQ, and cytokines were measured by immunoassay or flow cytometry. Transcriptome sequencing was performed on RNA-IC-stimulated pDCs from healthy individuals to assess the effect of IRAK4i and HCQ.

Results: In healthy individuals, RNA-IC induced interferon (IFN)-α, tumor necrosis factor (TNF)-α, IL-6, IL-8, IFN-γ, macrophage inflammatory protein (MIP)1-α, and MIP1-β production in pDC and NK cell cocultures. IFN-α production was selective for pDCs, whereas both pDCs and NK cells produced TNF-α. IRAK4i reduced the pDC and NK cell-derived cytokine production by 74-95%. HCQ interfered with cytokine production in pDCs but not in NK cells. In monocyte-depleted PBMCs, IRAK4i blocked cytokine production more efficiently than HCQ. Following RNA-IC activation of pDCs, 975 differentially expressed genes were observed (false discovery rate (FDR) < 0.05), with many connected to cytokine pathways, cell regulation, and apoptosis. IRAK4i altered the expression of a larger number of RNA-IC-induced genes than did HCQ (492 versus 65 genes).

Conclusions: The IRAK4i I92 exhibits a broader inhibitory effect than HCQ on proinflammatory pathways triggered by RNA-IC, suggesting IRAK4 inhibition as a therapeutic option in SLE.

Keywords: HCQ; IRAK4; NK; SLE; pDC.

Fig. 1

Regulatory effect of hydroxychloroquine (HCQ) and the interleukin-1 receptor associated kinase 4 inhibitor (IRAK4i) I92 on tumor necrosis factor (TNF)-α and interferon (IFN)-α production. a–d Cocultures of plasmacytoid dendritic cells (pDC) and natural killer (NK) cells from healthy donors were stimulated with RNA-containing immune complexes (RNA-IC) for 5 h (a,b) or 9 h (c,d) in the absence or presence of HCQ or I92. The frequencies of TNF-α- and IFN-α-producing NK cells (blue) and pDCs (red) were determined by flow cytometry. The dot plots represent one representative individual donor from two (HCQ) and four (IRAK4i) donors analyzed. e,f pDCs or NK cells were cultivated separately or in coculture in the presence of RNA-IC with or without I92 or HCQ. Cytokine levels were measured by immunoassays after 20 h. No IFN-α is produced by NK cells (data not shown). No cytokines were detected in the cell cultures in the absence of RNA-IC. Bars represent the mean with standard error of the mean (SEM) of nine donors from at least three independent experiments. Friedman’s test, uncorrected Dunn’s test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. BDCA blood dendritic cell antigen

Fig. 2

The interleukin-1 receptor associated kinase 4 inhibitor (IRAK4i) I92 displays a more prominent inhibitory effect than hydroxychloroquine (HCQ) on the production of proinflammatory cytokines in plasmacytoid dendritic cells (pDC) and natural killer (NK) cell cocultures and in NK cells alone. pDCs and NK cells from healthy donors were cultivated separately or in coculture in the presence of RNA-containing immune complexes (RNA-IC), with or without I92 or HCQ. The levels of a interleukin (IL)-6 and IL-8, and b interferon (IFN)-γ, macrophage inflammatory protein (MIP)1-α, and MIP1-β were measured by immunoassays after 20 h. Bars represent the mean with standard error of the mean (SEM) of nine donors from at least three independent experiments. Friedman’s test, uncorrected Dunn’s test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

The interleukin-1 receptor associated kinase 4 inhibitor (IRAK4i) I92 reduces both a tumor necrosis factor (TNF)-α and b interferon (IFN)-α production by monocyte-depleted peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients and healthy controls. The cells were stimulated with RNA-containing immune complexes (RNA-IC) in the presence or absence of I92 or hydroxychloroquine (HCQ), or were mock stimulated. TNF-α and IFN-α production in cell cultures was measured after 20 h by immunoassays. Box plots show median with interquartile range, based on 10–15 donors, in at least 10 independent experiments. Friedman’s test, uncorrected Dunn’s test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

The interleukin-1 receptor associated kinase 4 inhibitor (IRAK4i) I92 exhibits a broader inhibitory effect than hydroxychloroquine (HCQ) on cytokine production by monocyte-depleted peripheral blood mononuclear cells (PBMCs) from a systemic lupus erythematosus (SLE) patients and b healthy controls. Monocyte-depleted PBMCs were stimulated with RNA-containing immune complexes (RNA-IC) in the presence or absence of I92 or HCQ. Levels of interleukin (IL)-6, interferon (IFN)-γ, macrophage inflammatory protein (MIP)1-α, and MIP1-β in the cell cultures were measured after 20 h by immunoassays. Box plots show median with interquartile range, based on 10–13 donors from 10 independent experiments. Friedman’s test, uncorrected Dunn’s test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Differential effects on gene expression in RNA-containing immune complex (RNA-IC)-stimulated plasmacytoid dendritic cells (pDCs) by the interleukin-1 receptor associated kinase 4 inhibitor (IRAK4i) I92 and hydroxychloroquine (HCQ). RNA-sequencing analysis was performed on RNA-IC-stimulated pDCs from healthy donors (n = 4), cultivated for 6 h in the presence or absence of I92 or HCQ. The heat map showing the mean log2 fold change values for the 975 activation signature genes from RNA-IC versus mock-stimulated cells (left), RNA-IC I92 versus RNA-IC (middle left), RNA-IC HCQ versus RNA-IC (middle right), and the difference between the two compounds RNA-IC I92 versus RNA-IC HCQ treated cultures (right)

Fig. 6

The effect of interleukin-1 receptor associated kinase 4 inhibitor (IRAK4i) I92 or hydroxychloroquine (HCQ) treatment on gene expression in plasmacytoid dendritic cells (pDCs) stimulated with RNA-containing immune complex (RNA-IC). RNA sequencing analysis was performed on RNA-IC-stimulated pDCs from healthy donors (n = 4), cultivated for 6 h in the presence or absence of I92 or HCQ. Volcano plots comparing false discovery rate (FDR) versus log2 fold change (FC) for genes from a RNA-IC-stimulated pDCs relative to mock-treated cells, b RNA-IC I92 relative to RNA-IC-treated cells, c RNA-IC HCQ relative to RNA-IC-treated cells, or d RNA-IC HCQ relative to RNA-IC I92-treated cells. Genes labeled in blue are statistically significantly different between the two treatments (FDR < 0.05)