Dysregulated Responsiveness of Circulating Dendritic Cells to Toll-Like Receptors in ANCA-Associated Vasculitis

Abstract

Objective: Dendritic cells (DCs) are critical effectors of innate and adaptive immunity playing crucial roles in autoimmune responses. We previously showed that blood DC numbers were reduced in autoimmune antineutrophil cytoplasmic autoantibody-associated vasculitis (AAV). Here, we assessed toll-like receptor (TLR) responsiveness of blood DCs from patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA).

Methods: Blood samples from healthy controls (HCs), GPA, or MPA patients, without treatment, during acute phase (AP) or remission phase (RP) were analyzed. Cytokine production by DCs and T cells was assessed on whole blood by flow cytometry after TLRs or polyclonal stimulation, respectively.

Results: We first showed that GPA and MPA are associated with a decreased blood DC number during AP. Conventional DCs (cDCs) from patients with GPA and MPA in AP exhibited a profound decrease of IL-12/IL-23p40 production after TLR3, 4, or 7/8 stimulation compared to patients in remission and HC, with a return to normal values in RP. TNFα secretion was also affected, with a decrease in cDCs from GPA patients in AP after TLR3 stimulation but an increase after TLR7/8 stimulation. By contrast, the responsiveness of plasmacytoid DCs to TLR7 and 9 was only marginally affected. Finally, we observed that IFNγ-producing CD4⁺ T cell frequency was significantly lower in AP-GPA patients than in HC.

Conclusion: We describe, for the first time, a dysregulated response to TLRs of circulating DCs in AAV patients mostly affecting cDCs that exhibit an unexpected reduced inflammatory cytokine secretion possibly contributing to an altered Th cell response.

Keywords: ANCA-associated vasculitis; IL-12/IL-23p40; dendritic cells; dysregulation; toll-like receptor.

Figure 1

Decreased number of dendritic cells (DCs) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). Conventional DCs (cDCs) and plasmacytoid DCs (pDCs) were enumerated by flow cytometry on whole blood samples from patients with antineutrophil cytoplasmic autoantibody-associated vasculitis and healthy control [HC: n = 14, GPA-acute phase (AP): n = 15, GPA-remission phase (RP): n = 10, MPA-AP: n = 9, MPA-RP: n = 5].

Figure 2

Altered production of IL-12/IL-23-p40 and TNFα in stimulated conventional dendritic cells (cCDs). Whole blood samples were incubated with medium or toll-like receptor (TLR) 2, 3, 4, 7/8, or 9 ligands for 3.5 h and then stained for identification of cDCs (HLA-DR⁺, Lin⁻, CD11c⁺, CD123⁻) together with intracellular cytokine production IL-12/IL-23p40 (A) and TNFα (B). Healthy control (HC): n = 14, granulomatosis with polyangiitis (GPA)-acute phase (AP): n = 15, GPA-remission phase (RP): n = 10, microscopic polyangiitis (MPA)-AP: n = 9, MPA-RP: n = 5.

Figure 3

Slight decrease of cytokine production by plasmacytoid dendritic cells (pDCs) in active antineutrophil cytoplasmic autoantibody-associated vasculitis. Whole blood samples were incubated with medium or toll-like receptor (TLR) 7/8 or 9 ligands for 3.5 h and then stained for identification of pDCs (HLA-DR⁺, lin⁻, CD11c⁻, CD123⁺) together with intracellular cytokine production TNFα (A), IFNα (B), and IL-12/IL-23p40 (C). Healthy control (HC): n = 14, granulomatosis with polyangiitis (GPA)-acute phase (AP): n = 15, GPA remission phase (RP): n = 10, microscopic polyangiitis (MPA)-AP: n = 9, MPA-RP: n = 5.

Figure 4

Cytokine production and T cell subsets in antineutrophil cytoplasmic autoantibody-associated vasculitis. Intracellular production of IFNγ (A) and IL-17 (B) was assessed by flow cytometry in whole blood (WB) in CD4⁺ and CD8⁺ T cells after PMA and ionomycin stimulation in healthy control (HC): n = 10, granulomatosis with polyangiitis (GPA)-acute phase (AP): n = 15, GPA-remission phase (RP): n = 10, microscopic polyangiitis (MPA)-AP: n = 9, MPA-RP: n = 5. Frequencies of T cell subsets in WB using flow cytometry (C,D). Gating strategy to identify naïve/memory T cell and Th subsets (C). T cell subsets in HC: n = 7, GPA-AP: n = 9, GPA-RP: n = 9, MPA-AP: n = 7, MPA-RP: n = 2 (D). Results are expressed as percentage of positive cells.