Insulin-like growth factor-1 endues monocytes with immune suppressive ability to inhibit inflammation in the intestine

Abstract

The pathogenesis of some chronic inflammation such as inflammatory bowel disease is unclear. Insulin-like growth factor-1 (IGF1) has active immune regulatory capability. This study aims to investigate into the mechanism by which IGF1 modulates the monocyte (Mo) properties to inhibit immune inflammation in the intestine. In this study, the production of IGF1 by intestinal epithelial cells was evaluated by real time RT-PCR and Western blotting. Mos were analyzed by flow cytometry. A mouse colitis model was created with trinitrobenzene sulfonic acid. The results showed that mouse IECs produced IGF1, which could be up regulated by exposure to CpG-ODN (CpG-oligodeoxynueleotides) in the culture. Culture the CpG-ODN-primed IEC cells and Mos or exposure of Mos to IGF1 in the culture induced the Mos to express IL-10. The IGF1-primed Mos showed the immune suppressive effect on inhibiting the immune inflammation in the mouse colon. In conclusion, the IGF1-primed Mos are capable of suppressing immune inflammation in the intestine.

Figure 1. Intestinal epithelial cells express IGF1.

Mouse intestinal epithelial cells (IEC, a cell line) were cultured in the presence of CpG-ODN (CpG, in short; 500 ng/ml) for 48 h. Naïve BALB/c mice or colitic mice (6 mice/group) were gavage-fed with saline or CpG (10 μg/mouse in 0.3 ml saline) daily for 6 days. The epithelia were scrapped from the colon of the mice. The samples were analyzed by RT-qPCR and Western blotting. (A) the bars indicate the mRNA levels of IGF1. (B) the Western blots indicate the IGF1 protein contents. The bars below the blots indicate the integrated density of the blots. TNBS: Colitic mice induced by TNBS. The data of bars are presented as mean ± SD. *, p < 0.01, compared with saline group. #, p < 0.01, compared with group ″0″. The data are representatives of 6 independent experiments.

Figure 2. Intestinal Mos express IGF1R. Lamina propria mononuclear cells (LPMC) were isolated from the naïve BALB/c mouse colon, stained with the indicated antibodies (denoted in each sub-panel) and analyzed by flow cytometry.

(A) the dot plots indicate the frequency of Mos in LPMC. (B) the gated cells of panel A were further analyzed; the cells of upper gate are IGF1 receptor positive Mos; the cells of the lower gate are IGF1 receptor negative Mos. (C–F) the histograms indicate the phenotypes of the IGF1 receptor positive Mos; the cytokine profile is denoted in each histogram. (G) the bars indicate the summarized data of C–F. (H–K) the histograms indicate the phenotypes of the IGF1 receptor negtative Mos; the cytokine profile is denoted in each histogram. (L) the bars indicate the summarized data of H–K. The data are a representative of 3 independent experiments.

Figure 3. IGF1 induces IL-10⁺ Mos.

CD14⁺ F4/80^- IGF1R⁺ Mos were isolated from the spleen. The cells were exposed to IGF1 (as denoted above each histogram) in the culture for 72 h and analyzed by flow cytometry and RT-qPCR. (A–D) the histograms indicate the frequency of IL-10⁺ Mos. (E) the bars indicate the summarized data of A–D. (F) the bars indicate the mRNA levels of IL-10. The data of bars are presented as mean ± SD. *, p < 0.01, compared with saline group. The data represent 3 independent experiments.

Figure 4. CpG induces IECs to produce IGF1 and induces IL-10⁺ Mos.

Naïve Mos were cultured with IEC in a Transwell system (IECs in the inserts; Mos in the basal chambers) at a ratio of 1:1 in the presence of CpG (the doses of PMA are denoted above each histogram). The Mos were analyzed by flow cytometry. (A–E) the histograns indicate the frequency of IL-10⁺ Mos. (F) the bars indicate the summarized data of the IL-10⁺ Mos (mean ± SD. *, p < 0.01, compared with the dose ″0″ group). Anti-IGF1 Ab = 100 ng/ml. The data represent 3 independent experiments.

Figure 5. Immune suppressor functions of IGF1-primed Mos.

(A) IGF1-conditioned Mos and Teff (CD4⁺ CD25^- T cells; labeled with CFSE) were cocultured at a ratio of 10⁴:10⁴/well in the presence of anti-CD3/CD28 for 6 days. The additional treatment was denoted above each histogram. (A–F) the histograms indicate the frequency of proliferative Teff. (G) the bars indicate the summarized data of A–F. (H) the Western blots indicate the results of PD-L1 RNAi of Mos. IL-10d: IL-10-deficient. PD-L1n: PD-L1-null. csiRNA-Mos: The Mos were treated with control siRNA. The data of G are presented as mean ± SD. *, p < 0.01, compared with group A. The data are a representative of 3 independent experiments.

Figure 6. IGF1-primed Mos inhibit inflammation in the intestine.

A TNBS colitis model was created with BALB/c mice. The IBD-associated parameters were evaluated with samples collected from the mice with the procedures described in the text. The additional treatments are denoted above each image. (A–E), representative colon histology images (Magnification: ×100). (F) the bars indicate the inflammatory scores. (G) the curves indicate the body weight changes. (H) the bars indicate the MPO levels in colon tissue. Mos-c: Mos were conditioned with IGF1. Mos-n: Naïve Mos. IGF1: Mice were injected (i.p.) with recombinant IGF1 (25 μg/mouse) on day 1 and day 3 respectively. The data of bars are presented as mean ± SD. *, p < 0.01, compared with the TNBS/saline group. Each group consists of 6 mice. Samples from individual mice were processed separately. The data are a representative of 6 independent experiments.