Loss of the interleukin-6 receptor causes immunodeficiency, atopy, and abnormal inflammatory responses

Abstract

IL-6 excess is central to the pathogenesis of multiple inflammatory conditions and is targeted in clinical practice by immunotherapy that blocks the IL-6 receptor encoded by IL6R We describe two patients with homozygous mutations in IL6R who presented with recurrent infections, abnormal acute-phase responses, elevated IgE, eczema, and eosinophilia. This study identifies a novel primary immunodeficiency, clarifying the contribution of IL-6 to the phenotype of patients with mutations in IL6ST, STAT3, and ZNF341, genes encoding different components of the IL-6 signaling pathway, and alerts us to the potential toxicity of drugs targeting the IL-6R.

Figure 1.

Skin lesions, pedigrees, genetic sequencing results, and IL-6R expression levels in two patients with immunodeficiency, atopy, and abnormal inflammatory responses. (A) Photographs of the skin lesions of P1 demonstrating the excoriation and skin-colored nontender subcutaneous nodules (top) and a typical abscess (bottom) with the notable lack of surrounding erythema. (B) P1 diagnostic laboratory measurements of CRP and neutrophil and eosinophil count measured during multiple infective episodes over a 3-yr period. Normal ranges: CRP <5 mg/liter, neutrophils 1.7–6.5 × 10⁹/liter, eosinophils 0.04–0.5 × 10⁹/liter. (C) Photograph of the excoriating eczematous dermatitis found on the lower extremities of P2. (D) CADD/MAF graph of the variants identified in the IL6R gene of P2, plotted with the variants in the populations extracted from the databases ExAC and gnomAD. (E) Plot of read depth and zygosity at 23,368 genome-wide autosomal SNV positions in P1’s WGS data. Red dots, homozygous SNV calls; blue dots, heterozygous SNV calls. (F and G) The pedigrees, dideoxy sequencing chromatograms, and IL-6R mutations of P1 (F) and P2 (G). (H) Schematic representation of the IL-6R protein together with the positions of the mutations identified in P1 and P2. (I) Left: Fluorescence histograms of IL-6R expression within the memory (CD45RO⁺) and naive (CD45RO⁻) populations of gated CD3⁺CD4⁺ T cells from P1. Right: Dot-plots of IL-6R and CD27 of gated CD3⁺CD4⁺ T cells from P1. (J) Fluorescence histograms of IL6R expression on CD4⁺ and CD8⁺ T cells and B cells (CD19⁺) of P2. HC, healthy control.

Figure 2.

STAT signaling responses with and without ectopic expression of IL-6R in two patients with IL6R mutations. (A) Left: Dot plots of GP130 and CD45RO of gated CD3⁺CD4⁺ T cells from P1 and two healthy controls (HCs). Right: Dot plots of gp130 and CD27 of gated CD3⁺CD4⁺ T cells from P1. (B) Fluorescence histograms of the indicated anti-pSTAT proteins’ expression in CD4⁺ T cells from healthy controls and P1 upon stimulation. (C) Fluorescence histograms of the indicated anti-pSTAT proteins’ expression in CD4⁺ T cells from healthy controls and P2. (D) Left: Western blot of pSTAT3, STAT3, and actin as a loading control in P1 and healthy control PBMCs stimulated with the indicated IL-6 concentration for 30 min. Right: Western blot of pSTAT3, STAT3, pSTAT1, STAT1, and tubulin as a loading control in patient and healthy controls. PBMCs stimulated with the indicated cytokine concentrations for 30 min. (E) Left: pSTAT3 in healthy controls’ and IL-6R–null (P1) CD4⁺ T cells, transfected with a vector expressing WT IL-6R at baseline and after IL-6 stimulation. Right: The overlaid fluorescence histograms of pSTAT3 and pSTAT1 in PBMCs from the IL-6R–null patient in WT-IL-6R transfected and untransfected cells stimulated with IL-6. (F) Bar graphs indicating relative mean fluorescence intensity (rMFI) of pSTAT1 and pSTAT3 upon IL-6 stimulation of HEK293 T cells transfected with WT IL-6R, p.I279N/p.H280P IL6-R, p.I279N IL6-R, p.H280P IL6-R, or an empty vector (EV). Representative histograms are shown for each condition. Data represent two replicates of three independent experiments (unpaired two-tailed Student’s t test done on mean of technical replicates; *, P < 0.05; **, P < 0.01). Error bars represent SEM.

Figure 3.

Immunophenotype of IL-6R–null patient. (A) Split dot plot visualizations of how gene expression varies in P1 and her healthy father (HP1) generated from analysis of single-cell RNA-seq data from PBMCs. Top: Lineage defining transcription factor expression in memory CD4⁺ T cells. Bottom: Expression of genes indicating state of B lymphocyte differentiation and immunoglobulin isotype class in B cells. The size of the dot corresponds to the percentage of cells expressing the gene, and the color represents the average expression level. (B) GATA3 mRNA levels in ex vivo PBMCs isolated from P1 and healthy controls (HCs), detected by real-time PCR. (C) Flow cytometry analysis of CD25⁺FOXP3⁺ cells of P1 and two healthy controls, gated on the CD3⁺CD4⁺CD45RO⁺CD127⁻ compartment. (D) Naive cells from healthy controls (n = 3) and P1 were cultured under the indicated Th17-inducing conditions for 6 d. IL-17A, RORC, GATA3, and TBX21 mRNA levels were detected by real-time PCR. (E) SOCS3 mRNA levels of naive cells from P1 and healthy controls activated by the indicated cytokines for 18 h. Error bars represent SEM.

Figure 4.

Immunophenotype of patients with IL6R mutations. (A) Percentages of IL-17A–producing (left) and IFNγ-producing (right) CD4⁺ T cells from healthy controls (HCs; n = 5), P1 (dark square), and P2 (blue circle) from two independent experiments. Black, data from experiment 1; blue, data from experiment 2. (B) Flow cytometry analysis of expression of markers found in pathological effector Th2 cells in CRTH2⁺ memory CD4⁺ T cells. Mann–Whitney U test; *, P < 0.05. (C) Flow cytometry analysis of blood CXCR5⁺ memory cTFHs in P1, P2, and healthy controls. (D) Flow cytometry assessment of B lymphocyte class switching and memory cell formation in P1, P2, and healthy controls and the healthy parents (HP). Error bars represent SEM.