Muscle glycome in idiopathic inflammatory myopathies: Impact in IL-6 production and disease prognosis

Abstract

Idiopathic inflammatory myopathies (IIM) are a group of chronic autoimmune diseases mainly affecting proximal muscles. Absence of meaningful prognostic factors in IIM has hindered new therapies development. Glycans are essential molecules that regulate immunological tolerance and consequently the onset of autoreactive immune response. We showed that muscle biopsies from patients with IIM revealed a deficiency in the glycosylation pathway resulting in loss of branched N-glycans. At diagnosis, this glycosignature predicted disease relapse and treatment refractoriness. Peripheral CD4⁺ T cells from active-disease patients shown a deficiency in branched N-glycans, linked to increased IL-6 production. Glycan supplementation, restoring homeostatic glycosylation profile, led to a decrease in IL-6 levels. This study highlights the biological and clinical importance of glycosylation in IIM immunopathogenesis, providing a potential mechanism for IL-6 production. This pinpoints muscle glycome as promising biomarker for personalized follow-up and a potential target for new therapies in a patients' subgroup with an ominous evolution.

Keywords: Disease; Glycobiology; Glycomics; Health sciences.

Graphical abstract

Figure 1

Altered N-glycosylation profile of muscle biopsies at diagnosis associates with disease course and response to therapy in patients with IIM (A) Schematic representation of the N-glycosylation pathway from high-mannose N-glycans (recognized by GNA lectin) toward more complex N-glycans, with β1,6-branching antennae (detected by L-PHA lectin). (B) Representative images of skeletal muscle from IIM and HC, low L-PHA, and high GNA reactivity in IIM biopsies (scale bar = 100 μm). (C) Qualitative score of GNA and L-PHA staining intensity from total tissue from IIM and HC biopsies. Analysis performed for 22 IIM and 11 HC cases. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (D) Distribution of IIM disease cases in high (≥50%) and low (<50%) GNA reactivity, at each cellular component (muscular or stromal). Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (E) Quantitative analysis of staining intensity in each cellular compartment (muscular or stromal) of IIM compared with HC. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (F) MGAT1 and MAN2A1 mRNA expression levels on total muscle FFPE biopsies from patients with IIM and HC individuals. Each dot represents one biological sample. Relative quantification (RQ) of mRNA levels is expressed as mean ± SD (Mann-Whitney t-test: ∗p value<0.05, ∗∗p value<0.01). (G) ROC curve for the GNA reactivity from patients with IIM with poor disease course (more than 1 flare) and its respective AUC and p value. (H) The predictive capacity of high GNA reactivity and other clinic-pathological parameters to distinguish poor disease prognosis (that have more than 1 flare). (I) GNA reactivity ROC curve, respective AUC and p value, for IIM patients’ refractory to treatment. (J) The predictive capacity of high GNA reactivity and other clinic-pathological parameters to distinguish non-responder patients (that do not respond to therapy). The error bars represent the standard deviation of the data.

Figure 2

Altered N-glycosylation profile of CD4+ T cells associates with increased activation and cytokine production in patients with IIM (A) Cell surface β1,6-branching N-glycans (L-PHA) and high-mannose (GNA) N-glycans of CD4⁺ T cells from IIM and HC peripheral blood, analyzed by flow cytometry. Levels of median fluorescence intensity (MFI) from patients with IIM were normalized for the average of healthy control MFI. Fold change ratio is represented as mean ± SD. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (B) MGAT1 and (C) MAN2A1 mRNA expression levels on peripheral CD3⁺ T cells from patients with IIM and HC individuals. Each dot represents one biological sample. Relative quantification (RQ) of mRNA levels is expressed as mean ± SD (Mann-Whitney t-test: ∗p value<0.05,∗∗p value<0.01). (D) Percentage of IL-6-producing CD4⁺ T cells in peripheral blood, gated on CD4⁺ T cells with high vs. low levels of L-PHA staining, from patients with IIM and HC individuals. Percentage of cells are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). (E) Levels of total IL-6-producing CD4⁺ T cells, analyzed by flow cytometry. Percentage of cells are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (F) Levels of total IL-4-producing CD4⁺ T cells, analyzed by flow cytometry. Percentage of cells are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney tests. (G) Serum IL-6 quantification by ELISA from IIM and HC. Levels are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). (H) Cell surface β1,6-branching N-glycans (L-PHA) of CD4⁺ T cells from IIM and HC peripheral blood after 24 h stimulated with 0.5 μg/mL anti-CD3, analyzed by flow cytometry. Levels are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (I) Percentage of activated (CD25 or CD69) CD4⁺ T cells from IIM and HC peripheral blood after 24 h stimulated with 0.5 μg/mL anti-CD3, analyzed by flow cytometry. Percentages are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). (J) Percentage of IL-6-producing CD4⁺ T cells from IIM and HC peripheral blood after 24 h stimulated with 0.5 μg/mL anti-CD3, analyzed by flow cytometry. Percentages are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). (K) Levels of IL-6 produced by CD3⁻ cells, analyzed by flow cytometry. Percentages are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (L) Median fluorescence intensity (MFI) of DC-SIGN and mannose receptor (MR) in DCs and macrophages from IIM and HC. Levels are expressed as mean ± SD. Unpaired one-tailed Mann-Whitney test (∗p value<0.05, ∗∗p value<0.01). The error bars represent the standard deviation of the data.

Figure 3

GlcNAc supplementation of fresh muscle biopsies from IIM patients attenuates pro-inflammatory immune response (A) Experimental outline of 150 mM N-acetylglucosamine (GlcNAc) supplementation of IIM biopsies for 72 h. Each patient biopsy was longitudinally cut into 2 similar portions, one for the supplementation (GlcNAc 150 mM) and other for control without supplementation (IIM). After incubation, cells were lysed for protein extraction and lectin blot analysis, together supernatant which was collected and released cytokines were analyzed by cytokine bead assay (CBA). (B) Adjusted values of intensity from L-PHA and GNA lectin blot of whole tissue cell lysate with or without GlcNAc supplementation. Normalized for loading control (actin). Levels are expressed as mean ± SD. Mann-Whitney tests (∗p value<0.05, ∗∗p value<0.01). (C) Fold change of cytokines secreted to the supernatant by IIM biopsies supplemented with 150 mM GlcNAc for 72 h, normalized by the respective basal condition. Levels of released cytokine were analyzed by cytokine bead assay (CBA) One-way ANOVA followed by Tukey’s post test.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Levels are expressed as mean ± SD.