Toll-like receptor 4-, 7-, and 8-activated myeloid cells from patients with X-linked agammaglobulinemia produce enhanced inflammatory cytokines

Abstract

Background: Bruton tyrosine kinase (BTK) is a component of signaling pathways downstream from Toll-like receptors (TLRs) 2, 4, 7, 8, and 9. Previous work in BTK-deficient mice, cell lines, and cultured cells from patients with X-linked agammaglobulinemia (XLA) suggested defective TLR-driven cytokine production.

Objective: We sought to compare TLR-4-, TLR-7-, and TLR-8-induced cytokine production of primary cells from patients with XLA with that seen in control cells.

Methods: PBMCs from patients with XLA, freshly isolated plasmacytoid dendritic cells, monocytes, and monocytoid dendritic cells were activated with TLR-4, TLR-7, and TLR-8 agonists. Signaling intermediates and intracellular and secreted cytokine levels were compared with those seen in control cells.

Results: Although TLR-4, TLR-7, and TLR-8 activation of nuclear factor κB and mitogen-activated protein kinase pathways in cells from patients with XLA and control cells were comparable, TLR-activated freshly isolated monocytes and monocytoid dendritic cells from patients with XLA produced significantly more TNF-α, IL-6, and IL-10 than control cells. TLR-7/8-activated plasmacytoid dendritic cells produced normal amounts of IFN-α. In murine models BTK regulates the degradation of Toll-IL-1 receptor domain-containing adaptor protein, terminating TLR-4-induced cytokine production. Although this might explain the heightened TLR-4-driven cytokine production we observed, Toll-IL-1 receptor domain-containing adaptor protein degradation is intact in cells from patients with XLA, excluding this explanation.

Conclusion: In contrast to previous studies with BTK-deficient mice, cell lines, and cultured cells from patients with XLA suggesting impaired TLR-driven cytokine production, these data suggest that BTK inhibits TLR-induced cytokine production in primary human cells.

FIG 1

A and B, IκB-α and MAPK p38 phosphorylation in PBMCs treated with increasing concentrations of LPS (Fig 1, A) or CL097 (Fig 1, B) for 5 minutes. C and D, Phosphorylation after either no treatment (NT), LPS (1 µg/mL), or CL097 (2.5 µg/mL) was quantified and represented graphically as a percentage of β-actin in control cells (solid bars, n = 5) and cells from patients with XLA (open bars, n = 8). E, Phosphorylation of the MAPKs ERK, p38, and JNK in PBMCs treated as above with TLR ligands for 0 to 60 minutes.

FIG 2

Cytokines secreted by control monocytes (solid circles) and those from patients with XLA (open circles) treated as above for 6 hours (A and B) or with LPS (1 µg/mL) or CL097 (2.5 µg/mL) or for 48 hours (C, D, and E). ns, Not significant. *P < .05, **P < .01, and ***P < .001.

FIG 3

A–C, Cytokine production by control mDCs (solid circles) and those from patients with XLA (open circles) treated with LPS (1 µg/mL) or CL097 (2.5 µg/mL) for 24 hours. D, IFN-α production by pDCs from control subjects (solid circles) and patients with XLA (open circles) treated with CL097 (2.5 µg/mL) or loxoribine (500 µmol/L) for 24 hours. ns, Not significant. *P < .05, **P < .01, and ***P < .001.

FIG 4

A, Degradation of TIRAP in PBMCs from control subjects (NL; solid bars, n = 10) and patients with XLA (open bars, n = 13) treated with LPS (1.0 µg/mL) for 30 or 60 minutes. B, Representative Western blot of PBMCs from control subjects (NL) and 1 patient with XLA treated with LPS (1 µg/mL) for up to 240 minutes. NT, No treatment.