Functional genetic polymorphisms in ILT3 are associated with decreased surface expression on dendritic cells and increased serum cytokines in lupus patients

Abstract

Objective: Hyperactivity of the type I interferon (IFN) pathway is involved in the pathogenesis of systemic lupus erythematosus (SLE). Immunoglobulin like transcript (ILT3) is an immunohibitory transmembrane molecule which is induced by type I IFNs. ILT3 is expressed by plasmacytoid dendritic cells (PDCs), monocytoid dendritic cells (MDCs), and monocytes/macrophages. Given the pathogenic role of IFN in SLE, we hypothesised that the IFN-induced immunosuppressive ILT3 receptor may be dysfunctional in human SLE.

Methods: 132 European-derived and 79 Hispanic-American SLE patients were genotyped for two coding-change single nucleotide polymorphisms (SNPs) predicted to interfere with protein folding in ILT3 (rs11540761 and rs1048801). 116 control DNA samples and sera from healthy controls were also studied. We detected associations between ILT3 genotype and serum cytokine profiles. ILT3 expression levels on PDCs and MDCs from 18 patients and 10 controls were studied by flow cytometry.

Results: The rs11540761 SNP in the extracellular region was associated with decreased cell surface expression of ILT3 on circulating MDCs and to a lesser extent PDCs in SLE patients. The cytoplasmically located rs1048801 SNP was not associated with a change in dendritic cells expression of ILT3. Both SNPs were significantly and independently associated with increased levels of serum type I IFN activity in SLE patients. The rs1048801 SNP was also associated with increased serum levels of TNF-α.

Conclusions: Loss-of-function polymorphisms in ILT3 are associated with increased inflammatory cytokine levels in SLE, supporting a biological role for ILT3 in SLE.

Figure 1

Diagram of immunoglobulin-like transcript 3 gene with single nucleotide polymorphism (SNP) locations indicated by coloured lines above the gene, yellow indicates an intronic SNP, green indicates a synonymous SNP, and red indicates a coding change SNP. Ig 1 and Ig 2, immunoglobulin-like domains 1 and 2; Stalk, short peptide sequence between TM and Ig 2; TM, transmembrane sequence.

Figure 2

Immunoglobulin-like transcript 3 (ILT3) expression levels on dendritic cells of healthy controls and systemic lupus erythematosus (SLE) patients. ILT3 expression levels on plasmacytoid dendritic cells (PDCs) and monocytoid dendritic cells (MDCs) was determined by flow cytometry as described in Materials and Methods. ILT3 levels on (A) PDCs and (B) MDCs are similar between healthy controls, SLE patients, and in SLE patient subgroups with high or low serum type I IFN activity. (C) Serum type I IFN activity of patients sampled for data in this figure. ILT3 levels on (D) PDCs and (E) MDCs vary between SLE patients when data is stratified by ILT3 genotype with the rs11540761 single nucleotide polymorphism associating with significantly lower ILT3 levels on MDCs (coding change affecting amino acid position 18, G(Ser) or T(Arg)). Genotypes are shown as allelic combinations, for ex. GG, homozygous for G allele; GT, heterozygous, etc. No subjects were homozygous for rs11540761 in the flow cytometry experiments. Specific fluorescent intensities (SFI) values equal the fold increase in geometric mean fluorescence intensities of cells incubated with anti-ILT3 IgG₁-PE to cells incubated with IgG₁-PE. Mean+SD are shown. SFI values were compared by Student’s unpaired t-test. N, number of subjects.

Figure 3

Serum type I IFN activity and TNF-α levels in systemic lupus erythematosus (SLE) patients in relation to immunoglobulin-like transcript 3 (ILT3) genotype. SLE patients were stratified by ILT3 genotype at rs11540761 (coding change affecting amino acid position 18, G(Ser) or T(Arg)) and by ILT3 rs1048801 (coding change affecting amino acid position 413, A(Arg) or G(Gln)). Genotypes are shown as allelic combinations, for ex. GG, homozygous for G allele, GT, heterozygous, etc. p Values calculated by using the Mann-Whitney U test. Cytokine data are shown as median+ interquartile range. N, number of subjects.

Figure 4

IFN-α production by TLR9-activated plasmacytoid dendritic cells (PDCs) treated with immunoglobulin-like transcript 3 (ILT3) and immunoglobulin like transcript 7 (ILT7) crosslinking Abs. PDCs were treated with Abs to ILT3 and ILT7, or with control IgG, activated with CpG Type A DNA plus IL-3, and IFN-α activity was assayed in supernatants collected 18 h later. Crosslinking ILT7, but not ILT3, inhibits IFN-α secretion by PDCs. Mean±SD of four experiments is shown.