MKL1 expressed in macrophages contributes to the development of murine colitis

Abstract

Mice deficient in the megakaryoblastic leukaemia 1 (Mkl1) gene experience less severe dextran sulphate sodium (DSS)-induced colitis, implying that Mkl1 plays a pathological role in inflammatory bowel disease (IBD). However, the contribution of Mkl1 to the development of colitis remains to be elucidated. The expression of Mkl1 is higher in the colonic lamina propria macrophages (LPMac) of DSS-treated mice than in those of control mice. Therefore, we established a transgenic mouse line that overexpresses human MKL1 (MKL1-Tg) specifically in cells of the monocyte/macrophage lineage, in order to investigate the potential role of macrophage MKL1 in the pathogenesis of colitis. MKL1-Tg mice displayed spontaneous colon shortening and rectal prolapse. Flow cytometric and quantitative RT-PCR analyses revealed that, in MKL1-Tg mice compared to littermate controls, the population of LPMac was decreased and had an altered inflammatory phenotype indicative of impaired anti-inflammatory properties, whereas bone marrow-derived macrophages from MKL1-Tg mice skewed towards M1 polarisation. In addition, MKL1-Tg mice had higher susceptibility to DSS-induced colitis than their littermate controls. These observations indicated that MKL1 crucially contributes to the development of colitis via the regulation of the function of macrophages, suggesting that it may be a potential therapeutic target for the prevention of IBD.

Figure 1

Perturbation of the LPMac phenotype was associated with upregulation of Mkl1 during experimental murine colitis. C57BL/6 J mice were treated with 3% DSS or control drinking water for 4 days. (A) Representative cytograms of Ly6C and MHC-II expression on CD45⁺CD11b⁺CD11c^{low to mid}CD64⁺ cells. We calculated the proportion (rate, %) of the cells in the red frames, designated LPMac. (B) The expression levels of Mkl1 in CD45⁺CD11b⁺CD11c^{low to mid} CD64⁺Ly6C⁺ LPMo and CD45⁺CD11b⁺CD11c^{low to mid} CD64⁺Ly6C^—MHC-II⁺ LPMac were determined by quantitative RT-PCR. All experiments were repeated 5 times and data are shown as mean ± standard deviation (SD). *P < 0.05.

Figure 2

Characterisation of MKL1-Tg mice. (A) Body weights of male MKL1-Tg mice and non-Tg littermates were measured monthly. (B) Representative photograph of whole colon from female MKL1-Tg mice and control littermates at 4 months after birth. Colon length was measured (n = 3 for each group). Data are shown as mean ± SD. *P < 0.05. (C) Representative photographs of the perianal region of male control and MKL1-Tg mice with rectal prolapse. (D) Representative histological image showing cryptitis (arrowheads) in H&E-stained colonic tissue of 8-month-old MKL1-Tg mice. (E) Representative immunohistochemical images showing CD68⁺ cells stained with rat anti-CD68 antibody and Alexa Fluor® 488-conjugated goat anti-rat IgG. Green cells represent CD68⁺ cells (arrowhead) infiltrating the colonic submucosal layer of MKL1-Tg mice.

Figure 3

MKL1 overexpression affected the inflammatory phenotype of LPMac. (A) Representative cytograms showing the Ly6C and MHC-II expression on CD45⁺CD11b⁺CD11c^{low to mid}CD64⁺ cells from 6-month-old MKL1-Tg and non-Tg littermates. We also calculated the proportions of LPMac. (B) The expression levels of Mrc1, Cd163, Il10ra, and Tgfbr2 in CD45⁺CD11b⁺CD11c^{low to mid}CD64⁺Ly6C^—MHC-II⁺ LPMac were determined by quantitative RT-PCR. (C) The concentration of IFN-γ, IL-4, IL-17, and IL-10 in supernatants from LPLs were measured by ELISA. All experiments were repeated 3 times and data are shown as mean ± SD. *P < 0.05.

Figure 4

MKL1 modulated macrophage polarisation. (A) BMDMs from MKL1-Tg mice and non-Tg littermates were treated with rIL-4 (20 ng/ml) for 24 hours. The expression of CD206 on CD11b⁺F4/80⁺ BMDMs (histograms) was evaluated by flow cytometry. We calculated and plotted the mean fluorescence intensity (MFI). BMDMs from MKL1-Tg mice and non-Tg littermates were either treated with rIL-4 (20 ng/ml), or rIFN-γ (100 ng/ml) + LPS (50 ng/ml) for 16 hours. The expression levels of (B) M2 markers Mrc1, Arg1, Chi3l3, and Retnla, and (C) M1 markers Nos2, Il12b, Tnfa, and Il6 were determined by quantitative RT-PCR. All experiments were repeated 3 times and data are shown as mean ± SD. *P < 0.05.

Figure 5

MKL1-Tg mice developed fulminant DSS-induced colitis. MKL1-Tg mice and non-Tg littermates (8 weeks old) received 3% DSS in their drinking water for 7 days. (A) Male body weights were measured on day 0, 1, 4, and 7, and are shown as the percentage of initial body weight. (B) Representative image of whole colons from female MKL1-Tg mice and control littermates. Colon length was measured (n = 3 for each group). (C) Representative histological images (low-power field) of Swiss-rolled whole colon stained with H&E. Regions specified with blue lines were affected by colitis. Positions of anuses are indicated by circles. Lesion area was measured and plotted (n = 5 for each group). (D) Representative histological images (high-power field) of the proximal region of the colon stained with H&E. We calculated the histological score (n = 5 for each group). (E) MKL1-Tg mice and non-Tg littermates received 3% DSS in the drinking water for 4 days. Representative cytograms showing Ly6C and MHC-II expression on the CD45⁺CD11b⁺CD11c^{low to mid} CD64⁺ cells. We calculated the proportions of LPMac. Experiments were repeated 3 times. Data are shown as mean ± SD. *P < 0.05.