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. 2014 May;7(3):589-601.
doi: 10.1038/mi.2013.78. Epub 2013 Oct 16.

Aluminum Enhances Inflammation and Decreases Mucosal Healing in Experimental Colitis in Mice

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Free PMC article

Aluminum Enhances Inflammation and Decreases Mucosal Healing in Experimental Colitis in Mice

G Pineton de Chambrun et al. Mucosal Immunol. .
Free PMC article

Abstract

The increasing incidence of inflammatory bowel diseases (IBDs) in developing countries has highlighted the critical role of environmental pollutants as causative factors in their pathophysiology. Despite its ubiquity and immune toxicity, the impact of aluminum in the gut is not known. This study aimed to evaluate the effects of environmentally relevant intoxication with aluminum in murine models of colitis and to explore the underlying mechanisms. Oral administration of aluminum worsened intestinal inflammation in mice with 2,4,6-trinitrobenzene sulfonic acid- and dextran sodium sulfate-induced colitis and chronic colitis in interleukin 10-negative (IL10(-/-)) mice. Aluminum increased the intensity and duration of macroscopic and histologic inflammation, colonic myeloperoxidase activity, inflammatory cytokines expression, and decreased the epithelial cell renewal compared with control animals. Under basal conditions, aluminum impaired intestinal barrier function. In vitro, aluminum induced granuloma formation and synergized with lipopolysaccharide to stimulate inflammatory cytokines expression by epithelial cells. Deleterious effects of aluminum on intestinal inflammation and mucosal repair strongly suggest that aluminum might be an environmental IBD risk factor.

Figures

Figure 1
Figure 1
Aluminum worsens 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. (a) C57BL6 mice (n=14 per group) were fed with aluminum citrate (AluCi) or aluminum phosphate (AluP) (1.5 mg of Al element kg−1 per day) or with phosphate buffer saline (PBS) for 31 days. At day 28, colitis was induced by intrarectal administration of TNBS. Four days after colitis induction, mice were euthanized and colitis parameters were assessed. (b) The macroscopic Wallace score was determined as described in Methods. (c, d) Histopathological changes in colon tissues were examined by May-Grünwald and Giemsa (MGG) staining and scoring was performed as described in Methods. (e) Myeloperoxidase (MPO) activity was measured in colonic lesions. Results are expressed as the percentage of variation compared with TNBS-treated mice.
Figure 2
Figure 2
Aluminum citrate (AluCi) worsens dextran sodium sulfate (DSS)-induced colitis. (a) BALB/C mice (n=10 per group) were treated with 2.5% DSS and AluCi (1.5 mg of Al element kg−1 per day) or phosphate buffer saline (PBS) for 7 days. Control mice treated with AluCi or PBS without DSS were followed for the same period (n=5 per group). (b) Survival of mice was monitored until day 7 after the start of DSS. (c) Body weight was scored daily. * means P<0.05 between DSS±PBS and DSS±AluCi. (d) A disease activity index that included body weight variation, the presence of blood in stools, and stool consistency was calculated at day 7. (e) Myeloperoxidase (MPO) activity was measured on colons harvested at day 7. (f) Histopathological changes of colonic tissues were examined by May-Grünwald and Giemsa (MGG) staining.
Figure 3
Figure 3
Aluminum phosphate (AluP) but not zinc phosphate (ZnP) worsens dextran sodium sulfate (DSS)-induced colitis. (a) BALB/C mice (n=10 per group) were treated with 2.5% DSS and with increasing doses of AluP (0.15, 1.5, and 15 mg of Al element kg−1 per day) or ZnP (1.5 mg of Zn element kg−1 per day) or phosphate buffer saline (PBS) for 9 days. (b) Body weight was scored daily. * means P<0.05 between DSS±PBS and DSS±AluP 1.5 and 15, § means P<0.05 between DSS±PBS and DSS±AluP 0.15. (c) A disease activity index that included body weight variation, the presence of blood in stools, and stool consistency was calculated at day 9.
Figure 4
Figure 4
Aluminum citrate (AluCi) aggravates chronic colitis in interleukin 10-negative (IL10−/−) mice. (a) Eight-week-old IL10−/− mice were treated with AluCi (1.5 mg of Al element kg−1 per day) in their drinking water (n=15) or with water only (n=14) for 49 days. (b) The five parameters of the murine endoscopic index of colitis severity (MEICS) were determined. (c) Mini-endoscopic images were done as described in Methods. (d, e) Histopathological changes in colon tissues were examined by May-Grünwald and Giemsa (MGG) staining and scoring was performed as described in Methods. (f) Myeloperoxidase (MPO) activity was measured in colonic lesions.
Figure 5
Figure 5
Aluminum citrate (AluCi) increases the expression of pro-inflammatory cytokines in colitis. Il1β, Il17a, and Nlrp3 mRNA levels from homogenized colons of (a) mice treated with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and phosphate buffer saline (PBS) or treated with TNBS and AluCi, (b) mice treated with dextran sodium sulfate (DSS) and PBS or treated with DSS and AluCi, and (c) interleukin 10-negative (IL10−/−) mice treated or not with AluCi.
Figure 6
Figure 6
Aluminum phosphate (AluP) stimulates the expression of interleukin 8 (IL8) and IL1β by intestinal epithelial cells and enhances their response to bacterial stimuli. (a) Caco-2 cells were incubated with increasing doses of AluP (from 10 to 100 μg of Al element  ml−1). qPCR assay on Caco-2 cell lysates showed a dose-dependent increase in IL8 and IL1β expression in the presence of aluminum. (b) Caco-2 cells were co-stimulated with increasing doses of AluP (from 10 to 100 μg ml−1) and lipopolysaccharide (LPS). qPCR assay on Caco-2 cell lysates showed a dose-dependent increase in IL8 and IL1β expression in the presence of aluminum compared with LPS stimulation alone. *P<0.05 vs. controls.
Figure 7
Figure 7
Aluminum citrate (AluCi) extends the duration of dextran sodium sulfate (DSS)-induced colitis. (a) C57BL6 mice (n=10–15 per group) were treated with 2% DSS for 7 days, followed by regular drinking water for 12 days. In parallel, mice were treated with AluCi (1.5 mg of Al element kg−1 per day) or phosphate buffer saline (PBS) once a day until the end of the experiment. (b) Body weight was scored at baseline, D7, D13, D15, and D19. * means P<0.05; ** means P<0.01 between DSS±PBS and DSS±AluCi. (c) A disease activity index that included body weight variation, the presence of blood in stools, and stool consistency was calculated at day 19. (d) Myeloperoxidase (MPO) was measured in colonic samples harvested on day 19. (e) Histopathological changes in the colon tissues were examined by May-Grünwald and Giemsa (MGG) staining and scoring of histopathology, as described in Methods.
Figure 8
Figure 8
Aluminum citrate (AluCi) decreases the mucosal repair. (a) C57BL6 mice were treated with 2% dextran sodium sulfate (DSS) for 7 days, followed by regular drinking water for 10 days. AluCi (1.5 mg of Al element kg−1 per day) and phosphate buffer saline (PBS) treatment were started only after DSS discontinuation on day 7. (b) Body weight was scored daily. * means P<0.05. (c) Colon length and weight were measured from mice euthanized on day 0, day 6, and day 10 after the start of aluminum treatment and the colon weight/size ratio was calculated.
Figure 9
Figure 9
Aluminum citrate (AluCi) inhibits epithelial cell proliferation in vivo and in vitro. C57BL6 mice were fed with AluCi or phosphate buffer saline (PBS) for 10 days after dextran sodium sulfate (DSS)-induced colitis. (a, b) Proliferating cell nuclear antigen (PCNA) immunostaining of colon sections (a) and its quantification (b) showed a decrease in epithelial cell proliferation in mice having received aluminum as compared with mice administered phosphate buffer saline (PBS). (c, d) Terminal transferase dUTP nick end labeling (TUNEL) immunostaining of the same colons (c) and its quantification (d) shows no significant difference in epithelial cell apoptosis. Caco-2 cells were incubated with increasing doses of AluCi (50 and 100 μg ml−1) for 5 days. (e) MTT assay showed a decrease in epithelial cell proliferation. (f) Lactate dehydrogenase (LDH) activity assay in the supernatants did not reveal any significant variation.
Figure 10
Figure 10
Aluminum alters intestinal barrier integrity and induces granuloma formation. C57BL6 mice were fed with aluminum phosphate (AluP) (1.5 mg of Al element kg−1 per day, n=9) or phosphate buffer saline (PBS) (n=4) for 4 weeks. Bacterial counts in mesenteric lymph nodes (MLN) and colon were determined after 4 weeks. (a) Bacterial counts in MLN were significantly higher in aluminum-treated mice than in PBS-treated mice. (b) RT-qPCR assay of homogenized colons showed a decrease in Ocln mRNA in mice treated with aluminum as compared with PBS-treated mice. (c) Bacterial count of specific strains in the colonic mucosa showed no difference between aluminum-treated and PBS-treated mice. (d) Human peripheral blood mononuclear cells (PBMCs) were incubated for 4 days with increasing doses of AluP (from 1 to 100 μg of Al element per well; 1.5 ml medium per well). Quantitative analysis of the number of granulomas showed a dose-dependent enhancement of granuloma number in response to aluminum. (e) Representative light microscopy pictures ( × 10) of the culture wells after 4 days of reaction revealed large multicellullar structures (granulomas) in the presence of aluminum. (f) Human PBMCs were incubated for 5 days with non-pathogenic E. coli K-12 strain DH5α, AIEC strain LF82 or BCG, alone or in the presence of a low dose of aluminum (5 ng of Al element  ml−1). The number of granulomas according to their size (Index 1, small size, Index 2, big size) was counted. CFU, colony forming unit.

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