SIRT2 deficiency modulates macrophage polarization and susceptibility to experimental colitis

Abstract

Background: SIRT2 belongs to a highly conserved family of NAD+-dependent deacylases, consisting of seven members (SIRT1-SIRT7), which vary in subcellular localizations and have substrates ranging from histones to transcription factors and enzymes. Recently SIRT2 was revealed to play an important role in inflammation, directly binding, deacetylating, and inhibiting the p65 subunit of NF-κB.

Methods: A Sirt2 deficient mouse line (Sirt2-/-) was generated by deleting exons 5-7, encoding part of the SIRT2 deacetylase domain, by homologous recombination. Age- and sex-matched Sirt2-/- and Sirt2+/+ littermate mice were subjected to dextran sulfate sodium (DSS)-induced colitis and analyzed for colitis susceptibility.

Results: Sirt2-/- mice displayed more severe clinical and histological manifestations after DSS colitis compared to wild type littermates. Notably, under basal condition, Sirt2 deficiency does not affect the basal phenotype and intestinal morphology Sirt2 deficiency, however, affects macrophage polarization, creating a pro-inflammatory milieu in the immune cells compartment.

Conclusion: These data confirm a protective role for SIRT2 against the development of inflammatory processes, pointing out a potential role for this sirtuin as a suppressor of colitis. In fact, SIRT2 deletion promotes inflammatory responses by increasing NF-κB acetylation and by reducing the M2-associated anti-inflammatory pathway. Finally, we speculate that the activation of SIRT2 may be a potential approach for the treatment of inflammatory bowel disease.

Figure 1. Generation and characterization of the Sirt2^−/− mouse model.

(A) Schematic representation of the gene targeting strategy for exons 5–7 of the Sirt2 gene. (B) Western blot analysis of SIRT2 expression in the liver, heart, and colon of Sirt2^+/+ and Sirt2^−/− mice. (C) Genotype and sex distributions of newborn mice summarized from 162 colonies. (D) Body weight and body composition of Sirt2^+/+ and Sirt2^−/− mice.

Figure 2. Sirt2^−/− and Sirt2^+/+ mice colons are morphologically similar under normal conditions.

(A and B) Representative images of hematoxylin/eosin (A) and F4/80 staining (B) of the colon in Sirt2^+/+ and Sirt2^−/− mice. Number of F4/80⁺ cells are shown (right panel of B). n = 3/group. Scale bar = 20 µm.

Figure 3. Sirt2^−/− mice are more sensitive to DSS-induced colitis compared to Sirt2^+/+ animals.

(A–D) Severity of DSS-induced colitis was determined by body weight change (A), rectal bleeding scores (B), intestinal permeability (C), and histological scores (D) in Sirt2^+/+ and Sirt2^−/− mice. n = 10/group. (E–H) Histological changes in the intestine of the DSS-treated Sirt2^+/+ and Sirt2^−/− mice. Representative images demonstrating the extension of erosion/ulceration (arrows and dashed line indicate the extent of ulceration) (E), inflammation severity (arrows indicate leukocytic infiltrate and follicular aggregates) (F), mural involvement (arrows indicate transmural infiltration) (G), and damage distribution (arrows indicate sites of damage) (H). Corresponding histological scores are shown (lower panels). n = 10/group. Scale bar = 20 µm. Results are expressed as the mean ± SEM. *P<0.05; **P<0.01; ***P<0.001.

Figure 4. CD4⁺CD69⁺ T cells are increased in mesenteric lymph nodes from Sirt2^−/− mice with DSS-induced colitis.

(A) Representative images of FACS analysis demonstrating TCRb⁺ cells (left), and their composition sorted by CD4 and CD69 staining (right). (B) The composition of MLNs is compared between Sirt2^+/+ and Sirt2^−/− mice; TCRb⁺, CD4⁺, CD4⁺CD69⁻, and CD4⁺CD69⁺ cells. n = 10/group. Results are expressed as the mean ± SEM. *P<0.05; **P<0.01; ***P<0.001.

Figure 5. Plasma cytokine levels and cytokine mRNA levels in the colon of Sirt2^−/− mice with DSS-induced colitis.

(A–C) Measurements of serum cytokine levels in Sirt2^+/+ (+/+) and Sirt2^−/− (−/−) mice after DSS-induced colitis; TNFα (A), IL1β (B), and IL6 (C). (D–E) Cytokine mRNA levels in the colon of Sirt2^+/+ and Sirt2^−/− mice before and after DSS-induced colitis. Tnfα (D), Il1β (E), and Il6 (F). (G–H) mRNA levels of the genes related to maintenance of intestinal permeability, Ocln (G), Cldn1 (H), Zo1(I) in Sirt2^+/+ and Sirt2^−/− mice with or without DSS treatment. n = 10/group (with DSS treatment); n = 5/group (without DSS treatment). Results are expressed as the mean ± SEM. *P<0.05; **P<0.01; ***P<0.001.

Figure 6. Increased mRNA levels of pro-inflammatory genes and decreased levels of anti-inflammatory genes in Sirt2^−/− mouse-derived BMDMs.

(A) Sirt2 mRNA levels in Sirt2^+/+ (+/+) and Sirt2^−/− (−/−) mice. (B and C) mRNA levels of pro-inflammatory cytokine genes (Il1β, Tnfα, Il6, Mcp1) (B) and anti-inflammatory genes (Il4r, Il10) (C) in BMDMs from Sirt2^+/+ and Sirt2^−/− mice under basal conditions. n = 3/group. (D) mRNA levels of pro-inflammatory genes in BMDMs from Sirt2^+/+ and Sirt2^−/− mice upon LPS treatment. (E) mRNA levels of Gata3, Arg1, and Cd11c in BMDMs from Sirt2^+/+ and Sirt2^−/− mice upon IL4 treatment. Results are expressed as the mean ± SEM. *P<0.05; **P<0.01; ***P<0.001.

Figure 7. p65 subunit of NF-κB is hyperacetylated in Sirt2-deficient BMDMs.

(A) Western blot analysis of the acetylated p65 subunit of NF-κB, and total- and phosphorylated-IKbα in BMDMs from Sirt2^+/+ and Sirt2^−/− mice upon LPS treatment.(B) Quantification of the differences observed in (A) were obtained using ImageJ software. Results are expressed as the mean ± SEM. *P<0.05; **P<0.01; ***P<0.001 and the statistical significance was calculated comparing each group to untreated (−LPS) Sirt2^+/+. N = 3/group.