Dual TNF-α/IL-12p40 Interference as a Strategy to Protect Against Colitis Based on miR-16 Precursors With Macrophage Targeting Vectors

Abstract

Cytokines are central components of the mucosal inflammatory responses that take place during the development of Crohn's disease. Cell-specific combination therapies against cytokines may lead to increased efficacy and even reduced side effects. Therefore, a colonic macrophage-specific therapy using miR-16 precursors that can target both TNF-α and IL-12p40 was tested for its efficacy in experimental colitic mice. Galactosylated low molecular weight chitosan (G-LMWC) associated with miR-16 precursors were intracolonically injected into mice. The cellular localization of miR-16 precursors was determined. The therapeutic effects and possible mechanism were further studied in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice. The results show that specific upregulation of miR-16 level in colonic macrophages significantly reduces TNF-α and IL-12p40 expression, which could suppress the associated mucosal inflammation and ultimately result in the relief of colitic symptoms. This strategy, based on the dual silencing of colonic macrophage-specific cytokines, represents a potential therapeutic approach that may be valuable for colitis therapy.

Figure 1

Identification of TNF-α and IL-12p40 as targets of miR-16. (a) Schematic description of conserved binding sites for miR-16 and the seed-recognizing sites are marked. The levels of miR-16 (b), TNF-α (c), and IL-12p40 (d) from primary peritoneal macrophages after transfection with pre/anti-miR-16 or scrambled pre/anti-miR-16. (e) Luciferase activity containing full length 3'UTR of mouse TNF-α/IL-12p40 in Raw 264.7 cells was assayed after transfection with pre/anti-miR-16 or scrambled pre/anti-miR-16. The parental luciferase plasmid and the binding site mutant plasmid (Mut) were also used. Values are expressed as the means (mean ± SEM). Five samples were analyzed per condition, and experiments were performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Cellular distribution of miRNA precursors and cellular quantification of miR-16, TNF-α, and IL-12p40 after G-LMWC&Cy3-miRNA precursors or G-LMWC&pre-miR-16 administration. (a) Frozen sections of the colons from healthy mice, colitic mice without any treatment, colitic mice with naked Cy3 miRNA precursors or colitic mice with G-LMWC&Cy3-miRNA precursors were stained with colonic macrophages markers (red, Cy3 miRNA precursor; green, F4/80; blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining). Scale bar, 50 μm. The inner lumen (abbreviated as L) and outer wall (abbreviated as O) was marked on the image to indicate the orientation of colon section. (b) Local magnification of the frozen colon sections from colitic mice with G-LMWC&Cy3-miRNA precursors (red, Cy3 miRNA precursor; green, F4/80; blue, DAPI nuclear staining). Scale bar, 10 μm. (c) Flow cytometry used to analysis lamina propria mononuclear cells from mice with different treatments. (d) The ratio of Cy3 miRNA precursor⁺ cells in F4/80⁺ cells. (e) The level of miR-16 in different types of colonic cells after naked pre-miR-16, G-LMWC&pre-miR-16, or G-LMWC&pre-scramble administration at the dose of 5 mg miRNA precursors/kg body. (f,g) The mRNA levels of TNF-α/IL-12p40 in isolated colonic macrophages and the concentrations of TNF-α/IL-12p40 in the supernatants of colonic macrophages with different treatments. Values are expressed as the means (mean ± SEM). n = 7–8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

G-LMWC&pre-miR-16 complexes effectively reduced the levels of colonic TNF-α/IL-12p40. G-LMWC&pre-miR-16 or G-LMWC&pre-scramble were injected into TNBS-induced colitic mice by intracolonic administration at the dose of 5 mg miRNA precursors/kg body. The levels of colonic miR-16 (a), TNF-α/IL-12p40 (b,c) in colitis mice were examined by qRT-PCR. The expressions of colonic TNF-α/IL-12p40 were determined by enzyme-linked immunosorbent assay kits (b,c) or by immunofluorescence staining (d). Scale bar, 100 μm. n = 7–8 mice per group; values are expressed as the means (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001. NS, no significant change.

Figure 4

G-LMWC&pre-miR-16 complexes ameliorated TNBS-induced colitis. The therapeutic effect of pre-miR-16 in TNBS colitis mice was observed through body weight changes (a), survival analysis (b), disease activity index (c), myeloperoxidase activity determination (d), and colon photographs (e). Colon sections at day 3 from TNBS colitic mice that received pre-miR-16 were examined by H&E staining and histopathological scoring (f,g). Scale bar, 100 μm. The levels of colonic inflammatory cytokines (IFN-γ, IL-1β, IL-6, IL-12p70, IL-17A, and IL-23) from colitis mice with G-LMWC&pre-miR-16 treatment was determined (h). n = 7–8 mice per group; values are expressed as the means (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

The overexpression of IL-12p40/TNF-α 3'UTR-deficient plasmids abrogated the regulating ability of miR-16 on the colonic IL-12p40/TNF-α. G-LMWC&pre-miR-16, G-LMWC&pre-scramble, G-LMWC&IL-12p40 plasmids, or G-LMWC&TNF-α plasmids were injected into mice 3 days prior to TNBS induction at the dose of 5 mg plasmid/kg body. At day 3 in colitis mice, the levels of colonic miR-16 (a) and TNF-α/IL-12p40 (b,c) were examined by qRT-PCR. The expressions of colonic TNF-α/IL-12p40 were determined by enzyme-linked immunosorbent assay kits (b,c) or by immunofluorescence staining (d). Scale bar, 100 μm. n = 7–8 mice per group; values are expressed as the means (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001. NS, no significant change.

Figure 6

G-LMWC&pre-miR-16 complexes protected against TNBS-induced colitis by inhibiting IL-12p40 and TNF-α expression. The effects of administering IL-12p40/TNF-α plasmids to colitic mice were observed by body weight changes (a), survival analysis (b), disease activity index (c), myeloperoxidase activity determination (d), and colon photographs (e). Colon sections were examined by H&E staining and histopathological scoring at day 3 from colitic mice that received G-LMWC&pre-miR-16 treatment (f,g). Scale bar, 100 μm. n = 7–8 mice per group; values are expressed as the means (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.01. NS, no significant change.

Figure 7

The influence of administration of IL-12p40/TNF-α plasmids on the expressions of colonic cytokines. The levels of colonic inflammatory cytokines (IFN-γ, IL-1β, IL-6, IL-12p70, IL-17A, and IL-23) were determined from colitis mice with G-LMWC&pre-miR-16 treatment. n = 7–8 mice per group; values are expressed as the means (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001.