Complement receptor 3 influences toll-like receptor 7/8-dependent inflammation: implications for autoimmune diseases characterized by antibody reactivity to ribonucleoproteins

Abstract

Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFα in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFα secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFα release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.

Figures

FIGURE 1.

LA1, a CR3 agonist, attenuates TNFα release by THP-1 cells stimulated with hY3, TLR7, and TLR8 agonists via the degradation of MyD88.A, TNFα levels measured in supernatants generated from THP-1 after exposure to either hY3 RNA transfection, a TLR7 agonist, and a TLR8 agonist (18 h, 37 °C) in the presence of LA1 (pretreatment of 30 min, concentration varied). B, lactate dehydrogenase (LDH) release relative to a positive control (a condition employing lysis using Triton X-100 (TritonX)) in supernatants from THP-1 cells stimulated with a TLR7/8 agonist for 3, 6, and 18 h with or without LA1. TNFα levels from THP-1 cells stimulated with hY3 (C) or R848 (D) in the presence or absence of LA1; an oligonucleotided-based TLR7/8/9 inhibitor (TLRinh); an anti-CD11b antibody; or isotype control. Bars represent mean pg/ml TNFα ± S.E. (A, n = 6; B, n = 4). E, immunoblot analysis of MyD88 in lysates generated from THP-1 cells stimulated with hY3 (left) or a TLR7 agonist (right) in the presence or absence of LA1 or a TLR7/8/9 inhibitor. α-Tubulin serves as a loading control. Bars represent the ratio of MyD88 to α-tubulin for each condition (mean value ±S.E. (n = 3). *, p < 0.05, **, p ≤ 0.005; ***, p ≤ 0.0005.

FIGURE 2.

The effect of LA1 on TLR signaling varies depending on the timing of administration. TNFα was measured in the supernatants generated from PMA-differentiated THP-1 cells (A) or human PBMC-derived macrophages (B) transfected with hY3 RNA either 30 min after (LA1pre) or 30 min prior to (LA1post) the addition of LA1. Bars represent the mean ± S.E. (A, n = 4; B, n = 7). *, p < 0.05; **, p ≤ 0.005.

FIGURE 3.

The discordant effect of LA1 on hY3 stimulation extends to agonists of TLR7 and TLR8 but not the IL-1 receptor.A, TNFα was measured in the supernatants generated from PMA-differentiated THP-1 exposed to a TLR7 agonist, a TLR8 agonist, or IL-1β either 30 min after (LA1 pre) or 30 min prior to (LA1 post) the addition of LA1. Bars represent mean ± S.E. (n = 4). B, immunoblot analysis of MyD88 from THP-1 cells with or without a TLR7/8 agonist or IL-1β stimulation in the absence and presence of LA1 (5 μm added 30 min post-stimuli). α-Tubulin serves as a loading control. Aggregate data from three experiments utilizing the ratio of MyD88 to α-tubulin are reported as described in Fig. 1. *, p < 0.05. ND, not detected. UT, untreated.

FIGURE 4.

The inhibitory effect of LA1 varies depending on carrier status for the ITGAM polymorphism R77H. TNFα was measured in the supernatants generated from non-carrier (open bars, n = 6) or variant carrier (shaded bars, n = 3) macrophages stimulated with R848 in the presence or absence of LA1. Bars represent mean pg/ml TNFα ± S.E. *, p < 0.05; **, p ≤ 0.005.