Pathogenic mechanisms involving the interplay between adipose tissue and auto-antibodies in rheumatoid arthritis

Abstract

We aimed to evaluate the association between adipose tissue (AT) dysfunction, autoimmunity, and disease activity in rheumatoid arthritis (RA). A cross-sectional study including 150 RA patients and 50 healthy donors and longitudinal study with 122 RA patients treated with anti-tumor necrosis factor (TNF)-α, anti-interleukin 6 receptor (IL6R) or anti-CD20 therapies for 6 months were carried out. In vitro experiments with human AT and adipocyte and macrophage cell lines were performed. A collagen-induced arthritis mouse model was developed. The insulin resistance and the altered adipocytokine profile were associated with disease activity, the presence of anti-citrullinated proteins anti-bodies (ACPAs), and worse response to therapy in RA. AT in the context of arthritis is characterized by an inflammatory state alongside the infiltration of macrophages and B/plasmatic cells, where ACPAs can have a direct impact, inducing inflammation and insulin resistance in macrophages and promoting a defective adipocyte differentiation, partially restored by biologicals.

Keywords: Health sciences; Immunology; Rheumatology.

Graphical abstract

Figure 1

Association between adipocytokine profile and insulin resistance, disease activity, subclinical atherosclerosis, and autoimmunity (A) Cytokines serum levels of RA patients compared with HDs. (B) Adipokines serum levels of RA patients compared with HDs. (C) Association studies of adipocytokines with disease activity. (D) Association studies of adipocytokines with insulin resistance. (E) Correlation analysis of adipocytokines with ACPAs titers. (F) Percentage of RA patients with subclinical atherosclerosis, association with visfatin serum levels and ROC analysis. These analysis were performed in 150 RA patients and 50 HDs. HDs: healthy donors; RA: rheumatoid arthritis; mod: moderate; TNF-α: tumor necrosis factor alpha; IL: interleukin; AdipoQ: adiponectin; ACPAs: anti-bodies to citrullinated protein antigens; CIMT: carotid intima media thickness; AUC: area under the curve. Significance was determined by t-test or Mann–Whitney rank-sum test for two independent groups. One-way ANOVA or Kruskall–Wallis tests were performed for multiple comparisons. Pearson correlation coefficients were determined (p < 0.05 was considered statistically significant). Data are represented as mean ± SEM.

Figure 2

Effects of the CIA development at both systemic and adipose tissue levels (A) Design of the study: control (n = 5) and CIA mice (n = 20). (B) Plasma levels of adipocytokines and insulin resistance in CIA mice (n = 20) compared with the control group (n = 5). (C) mRNA relative expression of genes involved insulin signal in gWAT of CIA (n = 20) and control group mice (n = 5). (D) mRNA relative expression of B cell markers in gWAT in CIA (n = 20) and control group mice (n = 5). (E) mRNA relative expression of markers of presence and polarization state of macrophages in gWAT of CIA (n = 20) and control group mice (n = 5). (F) B cell and macrophage presence by immunohistochemistry of gWAT in CIA (n = 10) and control group mice (n = 5). (G) Adipokine proteome profile in gWAT of CIA (n = 5) and control group mice (n = 5). CIA: collagen induced arthritis; gWAT: gonadal white adipose tissue; TNF-α: tumor necrosis factor alpha; MCP-1: monocyte chemotactic protein 1; CD: cluster differentiation; IRS: insulin receptor substrate; GLUT-4: glucose transporter type 4; adipoQ: adiponectin; HOMA-IR: homeostatic model assessment-insulin resistance; LCN-2: lipocalin-2; CRP: C-reactive protein; IGFBP: insulin like growth factor binding protein; DPP-4: dipeptidylpeptidase 4; AHSG: alpha 2-HS glycoprotein; PTX-3: pentraxin 3; SERPINE-1: serpin peptidase inhibitor, clade E member 1; LEP: leptin; TIMP-1: tissue inhibitor of metalloproteinase 1; LIF: leukemia inhibitory factor; DLK-1: delta like non-canonical notch ligand 1; ESM-1: endothelial cell specific molecule 1; FGF-21: fibroblast growth factor 21; IL-6: interleukin-6; MOK: MAPK/MAK/MRK overlapping kinase; H-E: hematoxylin-eosin. Significance was determined by t-test or Mann–Whitney rank-sum test for unpaired groups. ∗Significant differences vs control group, p < 0.05. Data are represented as mean ± SEM.

Figure 3

Direct in vitro effect of IgG-ACPAs on human visceral adipose tissue, adipocytes, and macrophages (A) Expression of genes related to inflammation, insulin signal and lipid metabolism in explants of human visceral adipose tissue treated with enriched IgG-ACPAs or IgG-NHS. The experiment was performed in duplicate and repeated 10 times. (B) Impact of enriched IgG-ACPAs in the differentiation process of fibroblast to adipocyte from day 0 to day 9: quantification of lipid content through oil red O and bodipy staining. (C) mRNA relative expression of genes involved in lipid accumulation, lipogenesis, and insulin signal in adipocytes treated with enriched IgG-ACPAs, IgG-NHS, or non-treated during the differentiation process. (D) mRNA relative expression of genes associated with M1 macrophage polarization state in macrophages treated with enriched IgG-ACPAs or IgG-NHS. IgG: immunoglobulin; NHS: normal human serum; ACPAs: anti-bodies to citrullinated protein antigens; IL: interleukin; MCP-1: monocyte chemotactic protein 1; TNF-α: tumor necrosis factor alpha; IRS: insulin receptor substrate; AKT: kinase protein B; GLUT-4: glucose transporter type 4; PPAR-γ: peroxisome proliferator activated receptor gamma; SREBP-1a: sterol regulatory element-binding transcription factor 1; IFN-γ: interferon gamma; ARG-1: arginase 1; DGAT: diacylglycerol o-acyltransferase 1; PLIN-1: perilipin-1. Significance was determined by t-test or Mann–Whitney rank-sum test for two independent groups. One-way ANOVA or Kruskall–Wallis tests were performed for multiple comparisons. The experiments with macrophages and adipocytes were performed in triplicate and repeated three times. ∗Significant differences vs IgG-NHS, p < 0.05; ^asignificant differences vs non-treated, p < 0.05; ^bsignificant differences vs IgG-NHS, p < 0.05. Data are represented as mean ± SEM. (E) mRNA relative expression of genes associated with M2 macrophage polarization state, insulin signal and lipid metabolism in macrophages treated with enriched IgG-ACPAs or IgG-NHS.

Figure 4

Effect of biological drugs. Insulin resistance influence in the therapy response (A) Δ DAS28 in insulin resistance RA patients compared with insulin sensitivity RA patients after six months of treatment with biologicals (n = 67). (B) HOMA-IR levels at baseline in responders and non-responder RA patients (n = 67). (C) Insulin levels at baseline in responder and non-responder RA patients (n = 67). (D) Glucose levels at baseline in responder and non-responder RA patients (n = 123). (E) Visfatin levels at baseline in responder and non-responder RA patients (n = 123). (F) Vaspin levels at baseline in responder and non-responder RA patients (n = 123). (G) Effect of anti-CD20 therapy on the ACPAs titers and the adipocytokine profile after six months of treatment (n = 56). (H) Effect of anti-TNF-α therapy on the ACPAs titers and the adipocytokine profile after six months of treatment (n = 45). (I) Effect of the anti-IL6R therapy on the ACPAs titers and the adipocytokine profile after six months of treatment (n = 22). DAS28: disease activity score 28; HOMA-IR: homeostatic model assessment-insulin resistance; IR: insulin resistance; IS: insulin sensitivity; ns: non-significant; ACPAs: anti-bodies to citrullinated protein antigens; Resp: responder; TNF-α: tumor necrosis factor alpha: IL: interleukin; IL-6R: interleukin 6 receptor; CD: cluster differentiation; ADIPOQ: adiponectin. Significance was determined by t-test or Mann–Whitney rank-sum test for unpaired groups. t-test or Wilcoxon matched-pairs signed rank test were used for paired groups. ∗Significant differences vs baseline, p < 0.05. ∗∗Significant differences vs baseline, p < 0.01. ∗∗∗Significant differences vs baseline, p < 0.001. Data are represented as mean ± SEM.

Figure 5

In vitro effect of biological treatments in the adipocyte differentiation and macrophage polarization (A) Impact of anti-TNF-α and anti-IL6R in the adipocyte differentiation blockage induced by Ig G-ACPAs: O Red oil staining and lipid content analyses. (B) mRNA relative expression of genes related to lipid accumulation, lipogenesis, and insulin signal in adipocytes at day 9 of differentiation treated with IgG-ACPAs combined with anti-TNF-α or anti-IL6R. (C) mRNA relative expression of genes related to M1 macrophage markers in Thp-1-derived macrophages treated with IgG-ACPAs combined with anti-TNF-α or anti-IL6R for 24 h. (D) mRNA relative expression of genes related to M2 macrophage markers, insulin signal, and lipid metabolism in Thp-1-derived macrophages treated with IgG-ACPAs combined with anti-TNF-α or anti-IL6R for 24h. IgG: immunoglobulin; ACPAs: anti-bodies to citrullinated protein antigens; DGAT: diacylglycerol o-acyltransferase 1; PPAR-γ: peroxisome proliferator activated receptor gamma; SREBP-1a: sterol regulatory element-binding transcription factor 1; GLUT-4: glucose transporter type 4; IFN-γ: interferon gamma; IL: interleukin; MCP-1: monocyte chemotactic protein 1; TNF-α: tumor necrosis factor alpha; IRS: insulin receptor substrate; AKT: kinase protein B; ARG-1: arginase 1; PLIN-1: perilipin-1. One-way ANOVA or Kruskall–Wallis tests were performed for multiple comparisons. Each experiment was performed in triplicate and repeated three times. ^aSignificant differences vs IgG-ACPAs, p < 0.05; ^bSignificant differences vs IgG-ACPAs + anti-IL6R, p < 0.05. Data are represented as mean ± SEM.