Macrophages are essential for CTGF-mediated adult β-cell proliferation after injury

Abstract

Objective: Promotion of endogenous β-cell mass expansion could facilitate regeneration in patients with diabetes. We discovered that the secreted protein CTGF (aka CCN2) promotes adult β-cell replication and mass regeneration after injury via increasing β-cell immaturity and shortening the replicative refractory period. However, the mechanism of CTGF-mediated β-cell proliferation is unknown. Here we focused on whether CTGF alters cells of the immune system to enhance β-cell replication.

Methods: Using mouse models for 50% β-cell ablation and conditional, β-cell-specific CTGF induction, we assessed changes in immune cell populations by performing immunolabeling and gene expression analyses. We tested the requirement for macrophages in CTGF-mediated β-cell proliferation via clodronate-based macrophage depletion.

Results: CTGF induction after 50% β-cell ablation increased both macrophages and T-cells in islets. An upregulation in the expression of several macrophage and T-cell chemoattractant genes was also observed in islets. Gene expression analyses suggest an increase in M1 and a decrease in M2 macrophage markers. Depletion of macrophages (without changes in T cell number) blocked CTGF-mediated β-cell proliferation and prevented the increase in β-cell immaturity.

Conclusions: Our data show that macrophages are critical for CTGF-mediated adult β-cell proliferation in the setting of partial β-cell ablation. This is the first study to link a specific β-cell proliferative factor with immune-mediated β-cell proliferation in a β-cell injury model.

Keywords: CTGF; Macrophages; Proliferation; Regeneration; T-cells; β-cell.

Figure 1

Experimental design for β-cell ablation and CTGF overexpression. (A) Experimental outline and cohorts. (B–E) H&E staining for immune cell detection adjacent to islets. Representative images of Control (B), CTGF (C), Ablation (D), and Ablation + CTGF (E) islets after 2 days of CTGF induction. Insets highlight small, dark and closely clustered nuclei that are indicative of immune cells (white arrows).

Figure 2

Alterations to immune cell populations by β-cell ablation and CTGF induction. (A) Total number of pancreatic CD45 + cells and (B) Proportion of islet-localized CD45-positive cells. (C–D) Representative images of Ablation (C) and Ablation + CTGF (D) islets after 2 days CTGF. Insulin (green), CD45 (red). (E) Total number of F4/80-positive cells and (F) Proportion of islet-localized macrophages. (G–H) Representative images of Ablation (G) and Ablation + CTGF (H) islets after 2 days CTGF. Insulin (green), F4/80 (red). (I) Total number of CD3-positive cells. (J–K) Representative images of Ablation (J) and Ablation + CTGF (K) islets after 2 days CTGF. Insulin (green), CD3 (red). Immune cells within the exocrine or endocrine compartments are demarked by red and white arrows, respectively. n = 4. *p < 0.05, **p < 0.01.

Figure 3

CTGF induces expression of genes involved in the innate (A) and adaptive (B) immune response. Gene expression analysis on whole islets using Taqman Universal PCR Mastermix. Islets isolated from animals with/without β-cell ablation ± CTGF induction for 2 days. All samples were run in duplicate. n = 4. * compared to Control, ^# compared to CTGF induction, ˆ compared to Ablation. *, ^#, ˆp < 0.05, **p < 0.01.

Figure 4

CTGF-mediated β-cell regeneration is dependent on macrophages. (A) Experimental outline and cohorts. Total number of (B) F4/80- and (C) CD3-positive cells. (D) β-cell proliferation. (E) Proportion of MafA+ (red bars) or MafA− (yellow bars) β-cells. (F) Model of potential mechanisms of CTGF mediated β-cell mass regeneration. After 50% β-cell ablation, macrophages enter the islet to remove dead β-cells. Upon CTGF induction, more macrophages are recruited to the islet, elevating the levels of a secreted factor that promotes β-cell permissiveness to the proliferative factor. The β-cell proliferative factor is: 1. CTGF itself, 2. other CTGF-induced proliferative factors, such as 5-HT, Integrin β1, or HGF, 3. elevated levels of a macrophage-derived factor, or 4. upon CTGF induction, the character of recruited macrophages is altered by CTGF (i.e. polarization, chemokine profile etc.) resulting in the secretion of CTGF-induced macrophage factor. n = 6 (B–C) ***p < 0.001, ****p < 0.0001. (D) ****p < 0.0001 comparing 2 vs 1,3. ^###p < 0.0001 comparing 4 vs 2. (E) *p < 0.05 comparing 4 vs 1,3. ****p < 0.0001 comparing 2 vs 1,3. ˆ ˆ ˆ p < 0.001 comparing 4 vs 2.