Inhibition of phosphodiesterase-4 attenuates murine ulcerative colitis through interference with mucosal immunity

Abstract

Background and purpose: Ulcerative colitis (UC) is an aetiologically refractory inflammatory disease, accompanied by dysfunction of the epithelial barrier and intestinal inflammation. Phosphodiesterase-4 (PDE4) serves as an intracellular proinflammatory enzyme, hydrolyzing and inactivating cAMP. Though PDE4 inhibitors have been approved for pulmonary and dermatological diseases, the role of PDE4 inhibition in modulating mucosal immunity in the intestine remains ill-defined. This study was designed to explore whether PDE4 inhibition by apremilast exerts protective effects in dextran sulfate sodium-induced murine UC.

Experimental approach: Intestinal inflammation and disease severity were evaluated by morphological, histopathological and biochemical assays, and in vivo imaging. Expression of inflammatory mediators, components of PDE4-mediated pathways in colon and macrophages were determined using quantitative real-time PCR, ELISA, Luminex assay, immunostaining, or western blotting, along with siRNA knockdown. Immune cells in mesenteric lymph nodes and colonic lamina propria were analysed by flow cytometry.

Key results: Apremilast attenuated clinical features of UC, suppressing microscopic colon damage, production of inflammatory mediators, oxidative stresses, and fibrosis. Apremilast also promoted epithelial barrier function and inhibited infiltration of immune cells into inflamed tissues, through decreasing expression of chemokines and chemokine receptors. Furthermore, in UC, PDE4A, PDE4B, and PDE4D were highly expressed in colon. Apremilast not only inhibited PDE4 isoform expression but also activated PKA-CREB and Epac-Rap1 pathways and subsequently suppressed MAPK, NF-κB, PI3K-mTOR, and JAK-STAT-SOCS3 activation.

Conclusion and implications: Inhibition of PDE4 by apremilast protected against UC, by interfering with mucosal immunity. These findings represent a promising strategy for regulating intestinal inflammation.

Figure 1

Apremilast ameliorated DSS‐induced inflammation and oxidative stress in colitic mice. Murine colitis was established with 3% DSS for 7 days and drinking water for the next 4 days. (a) Body weight expressed as percentage of initial weight and disease activity index (DAI) mean values assigned based on the criteria described in Table 1. (b) Spleen index calculated by spleen weight (mg)/body weight (g). (c) Colon length. (d) Representative colon images. (e) Serum biochemical indices including ALB, ALP, TG, and TC. (f) Serum cytokine secretion. (g) Histological scores assigned. (h) Representative histological sections of colonic mucosa stained with haematoxylin and eosin (20× and 40× magnification). (i) Serum MPO, SOD, and MDA. (j) Colonic MPO, SOD, and MDA. (k) Flow cytometry analysis and quantification of ROS production in spleen and mesenteric lymph node cells. Data shown are means ± SEM; n = 8 mice per group. *P < 0.05, significantly different from vehicle (DSS only) group

Figure 2

Apremilast suppressed the production of inflammatory mediators and colonic fibrosis in colitic mice. (a) Cytokine production profile in the full‐thickness colon culture. (b) Protein levels of cytokines in the tissue homogenates. (c) The mRNA expression of cytokines in colon. (d) The mRNA expression of iNOS and COX‐2 in colon. (e) The mRNA expression of inflammasome‐related genes in colon. (f) Representative sections and quantification with positive area (%) of colonic mucosa with Masson staining (100× magnification). (g) The mRNA expression of fibrosis‐related genes in colon. Data shown are means ± SEM; n = 8 mice per group. *P < 0.05, significantly different from vehicle (DSS only) group

Figure 3

Apremilast protected the intestinal epithelial barrier function and prevented cytokine‐induced epithelial barrier disruption. (a) Bioluminescent imaging with L‐012 sodium was obtained under isoflurane anaesthesia using an IVIS Spectrum CT system. (b) Fluorescence imaging with FITC‐dextran administration (left) and serum fluorescence intensity of FITC‐dextran (right) were measured. (c) The expression of tight junction‐associated proteins (ZO‐1, E‐cadherin, and occludin) detected by western blot and α‐tubulin was used as a loading control. (d) Colonic tissues were immunofluorescently stained for ZO‐1 and E‐cadherin, and the nuclei were stained with DAPI. (e) The mRNA level of tight junction‐associated proteins. (f) The mRNA expression of MMP2, MMP3, and MMP9. (g) Barrier function was measured as TEER in Caco‐2 cell monolayers primed by TNF‐α and IFN‐γ. (h) FITC‐dextran permeability in cytokine‐induced Caco‐2 cells. (i) Quantification of fluorescence intensity of ZO‐1 in Caco‐2 cells. (j) Representative image of immunofluorescent staining for ZO‐1 in Caco‐2 cells. Data shown are means ± SEM. (a–f), n = 8 per group. *P < 0.05, significantly different from vehicle (DSS only) group. (g–j), n = 5. *P < 0.05, significantly different from TNF‐α plus IFN‐γ‐treated group

Figure 4

Apremilast regulated the leukocyte populations of mesenteric lymph nodes and lamina propria by suppressing the expression of chemokines and their receptors. (a) The percentage of macrophages (CD11b⁺F4/80⁺), neutrophils (CD11b⁺Gr‐1⁺), and dendritic cells (CD11b⁺CD11c⁺) in MLNs. (b) The percentage of naïve T cells (CD44⁻CD62L⁺, gated on CD3⁺CD4⁺), effector T cells (CD44⁺CD62L⁻, gated on CD3⁺CD4⁺), Th17 cells (CD4⁺IL‐17⁺, gated on CD3⁺), and Treg cells (CD25⁺Foxp3⁺, gated on CD3⁺CD4⁺) in MLNs. (c) The percentage of CD11b⁺ monocytes, macrophages, neutrophils, and dendritic cells in lamina propria. (d) The percentage of γδTCR⁺ T cells, CD4⁺ T cells, and CD8⁺ T cells in lamina propria. (e) Colonic tissues were immunofluorescence stained with CD11b, F4/80, and Ly6G, and the nuclei were stained with DAPI. (f) Colonic tissues were immunofluorescently stained for CXCR3 and CCR5. (g) The mRNA expression of chemokines and receptors in colon tissue. Data are shown as the representative images under flow cytometry and immunofluorescent staining. The summary data are shown as means ± SEM; n = 8 per group. *P < 0.05, significantly different from vehicle (DSS only) group

Figure 5

Apremilast inhibited lymphocyte proliferation and cytokine production upon ex vivo stimulation. CD4⁺ T cells were purified from MLNs and treated with anti‐CD3 plus anti‐CD28 antibodies to determine the CD4⁺ T cell proliferation (a) and (b) production of cytokines (IFN‐γ, IL‐2, IL‐10, and IL‐17A). (c) The proliferation of MLN cells treated with anti‐CD3 antibodies. and cytokine production (d) were determined. (e) The proliferation of MLN cells treated with LPS and (f) cytokine production. Data shown are means ± SEM; n = 8 mice per group. *P < 0.05, significantly different from vehicle (DSS only) group

Figure 6

Apremilast inhibited PDE4 expression, activated PKA–CREB and Epac‐Rap1 signalling, and subsequently interfered with JAK–STAT–SOCS3, PI3K–mTOR, NF‐κB, and MAPK pathways. (a) The mRNA expression of the isoforms of PDE4 in colonic tissue. (b) The expression of the isoforms of PDE4 and PKA–CREB signalling‐associated proteins. (c) Immunohistochemical staining for PDE4D, p‐p65, and p‐REK of colonic tissue. (d) Immunofluorescent staining for Epac1 in colonic tissue. Western blot analysis of JAK–STAT–SOCS3 signalling (e), NF‐κB‐ and MAPK‐mediated signalling (f) and PI3K–mTOR signalling (g). Data shown are representative images from western blot assays and from immunohistochemical staining. The summary data are shown as means ± SEM; n = 8 per group. *P < 0.05, significantly different from vehicle (DSS only) group

Figure 7

Apremilast suppressed the inflammatory responses in macrophages through PKA–CREB signalling. (a) TNF‐α and IL‐10 production in LPS‐stimulated BMDMs and RAW264.7 cells. (b) The phosphorylation of CREB and ATF‐1, following apremilast treatment, determined by western blot. (c) Immunofluorescent staining for phospho‐CREB upon apremilast treatment. BMDMs and RAW264.7 cells were treated with 10‐μM apremilast, 10‐μM H89 (PKA inhibitor), and 10‐μM Forskolin, cAMP level (d), phosphorylation of CREB and ATF‐1 (upper e, upper f, and g) were measured and TNF‐α production were determined upon LPS stimulation (h). BMDMs and RAW264.7 cells were transfected with PKA siRNA and NC SiRNA; then cells were treated with apremilast, H89, and forskolin. The phosphorylation of CREB and ATF‐1 (lower part of e; lower part of f) and TNF‐α (i) were measured. The summary data are shown as means ± SEM of three independent experiments

Figure 8

Diagram of how mucosal immunity in the DSS‐induced murine model of UC could be affected by apremilast to attenuate murine UC. PAMPs, pathogen‐associated molecular patterns; DAMPs, damage‐associated molecular patterns