Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical regulator of inflammatory signalling through toll-like receptors 4 and 7/8 in murine and human lungs

Abstract

Background and purpose: Toll-like receptors 4 (TLR4) and TLR7/TLR8 play an important role in mediating the inflammatory effects of bacterial and viral pathogens. Interleukin-1 receptor-associated kinase 4 (IRAK4) is an important regulator of signalling by toll-like receptor (TLR) and hence is a potential therapeutic target in diseases characterized by increased lung inflammatory signalling.

Experimental approach: We used an established murine model of acute lung inflammation, and studied human lung tissue ex vivo, to investigate the effects of inhibiting IRAK4 on lung inflammatory pathways.

Key results: We show that TLR4 stimulation produces an inflammatory response characterized by neutrophil influx and tumour necrosis factor-α (TNF-α) production in murine lungs and that these responses are markedly reduced in IRAK4 kinase-dead mice. In addition, we characterize a novel selective IRAK4 inhibitor, BI1543673, and show that this compound can reduce lipopolysaccharide (LPS)-induced airway inflammation in wild-type mice. Additionally, BI1543673 reduced inflammatory responses to both TLR4 and TLR7/8 stimulation in human lung tissue studied ex vivo.

Conclusion and implications: These data demonstrate a key role for IRAK4 signalling in lung inflammation and suggest that IRAK4 inhibition has potential utility to treat lung diseases characterized by inflammatory responses driven through TLR4 and TLR7/8.

Keywords: IRAK4; chronic obstructive pulmonary disease; exacerbations; interstitial lung disease; lung inflammation; toll‐ like receptors.

Figure 1

Lipopolysaccharide (LPS)-induced lung inflammation in interleukin-1 receptor-associated kinase 4 (IRAK4) wild-type and IRAK4 kinase-dead mice (IRAK4^D329A/D329A). LPS or phosphate-buffered saline (PBS) was administered via inhalation, and neutrophil numbers (upper panel) and tumour necrosis factor-α (TNF-α) concentration (lower panel) were measured in bronchoalveolar lavage fluid 4 h after LPS challenge. Data are given as mean ± SEM from 10 animals per group (5 male and 5 female).

Figure 2

Cigarette smoke-induced lung inflammation in interleukin-1 receptor-associated kinase 4 (IRAK4) wild-type and IRAK4 kinase-dead mice (IRAK4^D329A/D329A). Animals were challenged with the smoke from five cigarettes a day for four consecutive days, and neutrophil numbers (upper panel) and tumour necrosis factor-α (TNF-α) concentration (lower panel) were measured in bronchoalveolar lavage fluid 18 h after the last smoke treatment. Control animals were treated with ambient air. Data are given as mean ± SEM from five animals per group. Statistics: unpaired t-test.

Figure 3

Inhibition of lipopolysaccharide (LPS)-induced lung inflammation by the interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor BI1543673 in IRAK4 wild-type mice. BI1543673 was administered orally 1 h before challenging the animals by LPS inhalation. Bronchoalveolar lavage fluid (BALF) was prepared 4 h after the LPS challenge, and the neutrophil numbers (left panel) and tumour necrosis factor-α (TNF-α) concentration (centre panel) were measured. Data are given as mean ± SEM from four animals (control group) and eight animals (all other groups). In addition, (right panel) whole blood samples from the animals were stimulated ex vivo with LPS, and the TNF-α production was measured. Data are given as mean ± SEM from two animals (phosphate-buffered saline [PBS]) and six animals (LPS) per group.

Figure 4

Lipopolysaccharide (LPS) and resiquimod stimulation for 48 h show dose-dependent increases in interleukin-6 (IL-6) and interleukin-8 (IL-8) production in human lung tissue explants. Tissue was stimulated with a range of doses of LPS or resiquimod, and cytokine levels in supernatants were determined by Luminex. (a) LPS-induced IL-8 levels (n = 6), (b) resiquimod-induced IL-8 levels (n = 6), (c) LPS-induced IL-6 levels (n = 7) and (d) resiquimod-induced IL-6 levels (n = 4). Log EC₅₀ values were −7.553, −5.664, −8.058 and −8.224, respectively. Data are normalized to basal levels, mean fold ± SEM.

Figure 5

Cytokine production in human lung explants in response to stimulation with lipopolysaccharide (LPS) (10 μM) or resiquimod (10 μM) after either 24- or 48-h stimulations. n = 4–14. (a) interleukin-8 (IL-8), (b) interleukin-6 (IL-6), (c) tumour necrosis factor-α (TNF-α), (d) monocyte chemoattractant protein-1 (MCP-1), (e) interleukin-1β (IL-1β) and (f) macrophage inflammatory protein-1α (MIP1α). Median (interquartile range [IQR]) responses are shown relative to basal (100%).

Figure 6

The interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor BI1543673 significantly inhibits cytokine production in human lung explants in response to resiquimod or lipopolysaccharide (LPS). Tissue was stimulated with 10-μM resiquimod or 10-μM LPS for 48 h ± IRAK4 inhibitor, and cytokine/chemokine levels in supernatants were determined by Luminex. Examples are shown for (a) resiquimod/tumour necrosis factor-α (TNF-α) (n = 6, IC₅₀ 0.14 μM), (b) resiquimod/IL-1β (n = 6, IC₅₀ 0.32 μM), (c) LPS/IL-1β (n = 6, IC₅₀ 0.75 μM) and (d) LPS/granulocyte–macrophage colony-stimulating factor (GM-CSF) (n = 6, IC₅₀ 2.30 μM). Data are normalized to the resiquimod or LPS samples for each donor (100% response), mean ± SEM.