Galectin-3 exacerbates autoimmune diabetes by limiting regulatory T cell differentiation and function

Abstract

Galectin-3, a β-galactoside-binding lectin, has been implicated in several inflammatory and autoimmune diseases. However, the significance of circulating Galectin-3 in type 1 diabetes (T1D) remains unclear. Here, we report that compared to healthy controls, patients with T1D and their first-degree relatives (FDRs) exhibited significantly increased serum Galectin-3 levels primarily produced and secreted by monocytes/macrophages. Pharmacological inhibition (TD139) as well as knockout of Galectin-3 gene both attenuated Galectin-3-mediated suppression of regulatory T cells (T_reg cells) and protected from insulitis and diabetes onset in NOD mice. Mechanistically, Galectin-3 bound to and activated lymphocyte activation gene 3 (LAG3), a receptor expressed on activated T cells, subsequently suppressing the MEK/ERK signaling pathway and thereby hindering T_reg cell differentiation and function. In summary, our study identifies Galectin-3 as a potential biomarker for T1D and suggests that TD139 holds promise as a therapeutic candidate for patients with T1D and high serum Galectin-3 levels.

Fig. 1.. Serum Galectin-3 is increased in patients with T1D and their FDRs.

(A) The concentration of fasting serum Galectin-3 in healthy controls (HC, n = 132), Ab⁻ FDR (n = 76), Ab⁺ FDR (n = 30), and patients with T1D (n = 234). Data are expressed as median ± range. (B) The mRNA expression levels of Galectin-3 in CD14⁺ monocytes isolated from patients with T1D and HC (n = 10), calculated using the 2^−△△Ct method. (C) The concentration of fasting serum LPS in HC and T1D groups (n = 56). Data are expressed as median ± range. (D) Correlation between serum levels of Galectin-3 and LPS in HC and patients with T1D. (E to J) The THP-1 cell line, THP-1–cell induced macrophages, and the RAW 264.7 cell line were treated with LPS (100 ng/ml) or vehicle for 24 hours. The mRNA expression levels of Galectin-3 [(E), (G), and (I)] and the protein concentration of Galectin-3 in the supernatant [(F), (H), and (J)] were measured. Data in (E) to (J) are representative of at least three independent experiments with similar results. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.. The expression of Galectin-3 in pancreatic islets is elevated at an early age in NOD mice.

(A) Dynamic circulating levels of Galectin-3 in NOD and their control BALB/c mice at different ages (n = 6). (B) The mRNA abundance of Galectin-3 in the pancreatic islets of NOD and BALB/c mice, expressed as an arbitrary unit after normalization for β-actin mRNA levels, relative to the levels of 4-week-old BALB/c mice (n = 4). (C) The content of Galectin-3 in the pancreatic islet lysates of NOD and BALB/c mice (n = 3). (D and E) Representative patterns and median fluorescence intensity (MFI) of Galectin-3 expressed by CD45⁺ leukocytes (D) and CD45⁺F4/80⁺CD11b⁺ macrophages (E) in pancreatic islets of 4- and 12-week-old NOD and BALB/c mice (n = 4). (F) Galectin-3 expression in pancreatic islet macrophages of NOD mice at different ages (n = 3 to 4) from the National Center for Biotechnology Information database (GSE141782). (G) Representative patterns and MFI of Galectin-3 expressed by insulin⁺ pancreatic islet β cells of 4- and 12-week-old NOD and BALB/c mice (n = 4). Data are expressed as mean ± SEM. ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. 4w, 4 weeks; 12w, 12 weeks.

Fig. 3.. Galectin-3 deficiency alleviates autoimmune destruction of pancreatic islet β cells and diabetes in NOD mice.

(A) Diabetes incidence in Galectin-3^+/+ NOD (n = 20) and Galectin-3^−/− NOD mice (n = 16) at different ages. (B) Glucose excursion curve after receiving intraperitoneal glucose tolerance test (IPGTT) and the quantification of area under the curve in IPGTT (n = 8 to 9). (C) Representative images of H&E analysis for pancreatic sections and (D) insulitis scores in 12-week-old female Galectin-3^+/+ NOD and Galectin-3^−/− NOD mice (n = 4). Scale bar, 50 μm. (E) Representative images of immunohistochemistry (IHC) staining of TUNEL (green) and insulin (red) in pancreas of 12-week-old female Galectin-3^+/+ NOD and Galectin-3^−/− NOD mice (n = 6). Scale bar, 20 μm, with magnification of ×400. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.. Galectin-3 deficiency enhances T_reg cell proportion and decreases perforin-expressing CD8⁺ T cells in pancreatic islets of NOD mice.

Pancreatic islets from 12-week-old nondiabetic Galectin-3^+/+ NOD and Galectin-3^−/− NOD mice were harvested and subject to flow cytometry analysis (n = 5). Frequencies of CD4⁺ and CD8⁺ T cells (A) and T_reg cells (CD4⁺CD25⁺FOXP3⁺) (B) are shown as representative fluorescence-activated cell sorting plots. (C to E) Frequencies of T_H1 (CD4⁺IFN-γ⁺) (C), T_H2 (CD4⁺IL-4⁺) (D), and T_H17 (CD4⁺IL-17⁺) (E) subsets among CD45⁺ cells in islets of 12-week-old Galectin-3^+/+ NOD and Galectin-3^−/− NOD mice. (F to J) Frequencies of perforin⁺CD8⁺ (F), Tc1 (CD8⁺IFN-γ⁺) (G), Tc2 (CD8⁺IL-4⁺) (H), Tc17 (CD8⁺IL-17⁺) (I), and granzyme B⁺CD8⁺ (J) subsets among CD45⁺ cells in islets of 12-week-old Galectin-3^+/+ NOD and Galectin-3^−/− NOD mice. Data are expressed as mean ± SEM. *P < 0.05.

Fig. 5.. Galectin-3 exerts negative effects on T_reg cell differentiation and function.

(A) Frequency of CD4⁺CD25⁺FOXP3⁺ T_reg cells after exposure of naïve CD4⁺ T cells to T_reg cell–inducing conditions with or without Galectin-3 recombinant protein at different concentrations (n = 6). (B) Frequency of Ki67⁺ cells after T_reg cell induction (n = 6). (C) Apoptosis of T_reg cells was determined by annexin V staining (n = 3). (D) The percentage of proliferated T_reg cells was defined by carboxyfluorescein diacetate succinimidyl ester assay after 72 hours of culture (n = 4 to 6). (E and F) Cytokine profile of culture supernatants of isolated T_reg cells (n = 4). (G) CD8⁺ T cells were stimulated with plate-coated anti-CD3/CD28 and cultured either alone or with Galectin-3–pretreated or vehicle-pretreated T_reg cells at the indicated ratios. Perforin expression in CD8⁺ T cells was measured by flow cytometry after 72 hours. (H) CD8⁺ T cells were cocultured with T_reg cells in a Transwell system in the presence or absence of Galectin-3 protein, IL-10, or TGF-β neutralizing antibodies. Perforin expression was measured by flow cytometry after 72 hours. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6.. LAG3 signaling mediates the suppressive effects of Galectin-3 on T_reg cell differentiation.

(A) Expression levels of candidate Galectin-3–binding receptors in T_reg cells (n = 3). (B) Expression of LAG3 was examined on T_reg cells after induction in different groups as indicated (n = 3 to 6). (C and D) Naïve CD4⁺ T cells purified from WT C57BL/6J splenocytes were cultured under T_reg cell–polarizing conditions in vitro, treated with Galectin-3 protein alone or in combination with LAG3 neutralizing antibody for 72 hours. The frequencies of CD4⁺CD25⁺FOXP3⁺ T_reg cells (C) as well as Ki67⁺ cells (D) were assessed by flow cytometry (n = 7). (E) Heatmap showing the expression of T_reg cell function–associated genes in control T_reg cells versus Galectin-3–treated T_reg cells (n = 3). (F) Volcano plot of DEGs between groups. Log₂ FC, log₂ fold change. (G) GSEA plot showing the enrichment of the MAPK signaling pathway in Galectin-3–treated T_reg cells compared to control T_reg cells. (H and I) Immunoblot analysis of MEK/ERK phosphorylation in T_reg cells stimulated with Galectin-3, with or without LAG3 blockade. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NES, normalized enrichment score; FDR, false discovery rate.

Fig. 7.. Pharmacological inhibition of Galectin-3 ameliorates spontaneous development of insulitis and diabetes in NOD mice.

(A) Schematic diagram showing the protocol of TD139 treatment. (B) Incidence of diabetes expressed as percentage of diabetic mice at different ages (n = 20). (C) Glucose excursion curve after receiving IPGTT (n = 8). (D) Serum insulin concentration at 0 and 15 min during the IPGTT in 12-week-old TD139 or vehicle-treated NOD mice (n = 7 to 8). (E) Representative H&E staining of pancreas from 12-week-old NOD mice treated with TD139 or vehicle (n = 4). (F) Insulitis scores calculated on the basis of histological evaluation of pancreatic section as in (E). (G) Frequencies of CD4⁺ and CD8⁺ T cells in total CD3⁺ T cells from islets of 12-week-old NOD mice treated with TD139 or vehicle (n = 4). (H) Frequencies of T_reg cells (CD4⁺CD25⁺FOXP3⁺) from islets of 12-week-old NOD mice treated with TD139 or vehicle (n = 4). (I to K) Frequencies of perforin⁺CD8⁺ T, Tc1 (CD8⁺IFN-γ⁺), Tc2 (CD8⁺IL-4⁺), Tc17 (CD8⁺IL-17⁺), and granzyme B⁺CD8⁺ T subsets from islets of 12-week-old NOD mice treated with TD139 or vehicle (n = 3 to 4). (L) Frequencies of T_H1 (CD4⁺IFN-γ⁺), T_H2 (CD4⁺IL-4⁺), and T_H17 (CD4⁺IL-17⁺) from islets of 12-week-old NOD mice treated with TD139 or vehicle (n = 4). Data are expressed as mean ± SEM. *P < 0.05 and **P < 0.01.