Annexin A1 regulates intestinal mucosal injury, inflammation, and repair

Abstract

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (-/-) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (-/-) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.

FIGURE 1

Increased expression of AnxA1 in colonic mucosal tissues following DSS treatment. Western blot analysis of mucosal lysates from nontreated and DSS-treated WT BALB/c animals (A) revealed increased AnxA1 protein. Based on densitometric analysis of Western blots from these studies (B), AnxA1 levels were increased 1.5-fold following DSS treatment (*, p-value <0.05). No AnxA1 expression was observed in mucosal lysates from AnxA1 (−/−) mice (C). Western blot for actin was used as loading control.

FIGURE 2

AnxA1 expression is increased in intestinal epithelial cells following DSS treatment. Immunofluorescence analysis of WT BALB/c mucosa revealed expression of AnxA1 in both crypt and surface intestinal epithelial cells (A; arrows) [Bar = 50 μm]. In surface enterocytes (B), AnxA1 localizes in the apical and lateral plasma membranes as well as in the cytoplasm, whereas in crypt epithelial cells it appears to be exclusively in the cytoplasm (C) [Bars = 20 μm]. Following DSS treatment, AnxA1 levels are increased in both surface and crypt epithelial cells compared with nontreated animals (B-E; images taken at identical detector gain settings) [Bars = 20 μm]. Integrated pixel intensity analysis (F) revealed a 1.7-fold increase in AnxA1 levels in intestinal epithelial cells after 7 days of DSS treatment (*, p-value <0.05). A secondary Ab control (G) and staining of AnxA1 (−/−) mucosa (H) demonstrated no tissue staining [Bars = 50 μm].

FIGURE 3

AnxA1-deficient mice exhibit increased susceptibility to DSS-induced acute colitis. AnxA1 (−/−) mice demonstrated a significantly higher weight loss compared with WT controls beginning at day 3 of DSS treatment (A; *, p-value <0.05, n = 7 mice). A sustained increase in the DAI in AnxA1 (−/−) mice compared with WT controls was observed on days 1–7 of DSS treatment (B; *, p-value <0.05, n = 7 mice). Rectal bleeding scores (C) were also increased in AnxA1 (−/−) animals compared with WT controls during DSS treatment (*, p-value <0.05, n = 7 mice). An increase in the colon weight/length ratio was observed in AnxA1 (−/−) mice compared with WT controls following 7 days of DSS administration (D; *, p-value <0.05, n = 7 mice).

FIGURE 4

AnxA1-deficient mice have more severe histopathologic injury in DSS-induced colitis. Representative photomicrographs of H&E-stained histologic sections (A) demonstrate more severe colonic injury in AnxA1 (−/−) animals compared with WT controls following 7 days of DSS treatment. Note the increased degree of epithelial injury, increased leukocyte infiltration, and involvement of submucosal tissues in AnxA1 (−/−) mice (inset, arrows). Histologic scoring of colonic injury (B) is significantly higher than that of WT DSS-treated animals (*, p-value <0.05, n = 7 mice). Colonic MPO activity (C) is also significantly higher in AnxA1 (−/−) mice compared with WT BALB/c animals following DSS treatment (*, p-value <0.05, n = 7 mice).

FIGURE 5

AnxA1-deficient mice exhibit impaired clinical and histopathologic improvement following withdrawal of DSS treatment. AnxA1 (−/−) mice exhibited a significant reduction in the rate of weight gain following withdrawal of DSS treatment compared with WT controls (A; *, p-value <0.05, n = 7 mice). Similarly, AnxA1 (−/−) animals showed an impairment in recovery following DSS withdrawal as determined by DAI (B; *, p-value <0.05, n = 7 mice). Representative photomicrographs of H&E-stained histologic sections (C) demonstrate more severe histopathologic injury following 7 days of DSS treatment in AnxA1 (−/−) animals compared with WT controls. After 7 days of recovery, WT animals appeared to exhibit slightly less epithelial injury and reduced inflammation. However, a mild degree of architectural irregularity as evidenced by minor irregular crypt spacing was identified. In contrast, while the inflammation in AnxA1 (−/−) animals was improved to a small extent following 7 days of recovery from DSS-induced colitis, significantly increased epithelial injury with the development of architectural irregularity was observed (C). Histologic scoring (D) of colonic injury revealed some improvement of histopathologic changes in WT animals, whereas an overall increase in injury was found in AnxA1 (−/−) animals (*, p-value <0.05, n = 7 mice). Measurement of MPO activity (E) did not reveal a statistically significant difference in leukocyte infiltration in WT animals between 7 days of DSS treatment and after 7 days of recovery. MPO activity in AnxA1-deficient animals improved to a degree similar to that of WT controls at day 7 of DSS treatment and was less than that of WT controls following a 7 day recovery period (*, p-value <0.05, n = 7 mice).

FIGURE 6

ALX/FPRL-1 stimulation rescues the susceptibility of AnxA1-deficient mice to DSS-induced colitis. AnxA1 (−/−) animals demonstrated a more pronounced weight loss following DSS treatment that was restored to that of WT controls with ALX/FPRL-1 stimulation via administration of 15-epi-lipoxin A4 (denoted as L in the figure) (A; *, p-value <0.05, n = 7 mice). Conversely, WT animals did not show a reduction in weight loss due to ALX/FPRL-1 stimulation during DSS treatment. AnxA1 (−/−) mice exhibit an increased DAI over the course of DSS treatment which was restored similar to that of WT controls on days 4–7 with simultaneous ALX/FPRL-1 agonization via 15-epi-lipoxin A4 treatment (B; *, p-value <0.05, n = 7 mice). Treatment of WT animals with 15-epi-lipoxin A4 did not results in a consistent decrease in the DAI during DSS administration. Representative photomicrographs of H&E-stained histologic sections (C) and histologic scoring (D; *, p-value <0.05, n = 7 mice) demonstrate a similar degree of DSS-induced colonic injury in AnxA1-deficient animals treated with 15-epi-lipoxin A4 and WT controls (*, p-value <0.05). 15-epi-lipoxin A4 did not significantly improve the histologic injury induced by DSS in WT BALB/c mice.

FIGURE 7

AnxA1 (−/−) mice exhibit reduced ALX/FPRL-1 expression following DSS treatment. Total RNA was extracted from mucosal tissues from control and DSS-treated WT and AnxA1 (−/−) mice and subjected to RT-PCR analysis for ALX/FPRL-1, FPR-rs2, and FPR1. Although ALX/FPRL-1 levels were suppressed in WT type animals treated with DSS, a further diminishment of ALX/FPRL-1 mRNA was observed in AnxA1 (−/−) animals following DSS treatment (A; n = 3 animals). FPR-rs2 expression did not change following DSS administration in WT animals but was dramatically reduced in AnxA1 (−/−) mice following a 7 course of DSS treatment (B; n = 3 animals). A similar reduction in FPR1 expression was observed following DSS treatment in WT and AnxA1 (−/−) animals (C; n = 3 animals).