Modulation of neutrophil motility by curcumin: implications for inflammatory bowel disease

Abstract

Background: Neutrophils (PMN) are the first cells recruited at the site of inflammation. They play a key role in the innate immune response by recognizing, ingesting, and eliminating pathogens and participate in the orientation of the adaptive immune responses. However, in inflammatory bowel disease (IBD) transepithelial neutrophil migration leads to an impaired epithelial barrier function, perpetuation of inflammation, and tissue destruction via oxidative and proteolytic damage. Curcumin (diferulolylmethane) displays a protective role in mouse models of IBD and in human ulcerative colitis, a phenomenon consistently accompanied by a reduced mucosal neutrophil infiltration.

Methods: We investigated the effect of curcumin on mouse and human neutrophil polarization and motility in vitro and in vivo.

Results: Curcumin attenuated lipopolysaccharide (LPS)-stimulated expression and secretion of macrophage inflammatory protein (MIP)-2, interleukin (IL)-1β, keratinocyte chemoattractant (KC), and MIP-1α in colonic epithelial cells (CECs) and in macrophages. Curcumin significantly inhibited PMN chemotaxis against MIP-2, KC, or against conditioned media from LPS-treated macrophages or CEC, a well as the IL-8-mediated chemotaxis of human neutrophils. At nontoxic concentrations, curcumin inhibited random neutrophil migration, suggesting a direct effect on neutrophil chemokinesis. Curcumin-mediated inhibition of PMN motility could be attributed to a downregulation of PI3K activity, AKT phosphorylation, and F-actin polymerization at the leading edge. The inhibitory effect of curcumin on neutrophil motility was further demonstrated in vivo in a model of aseptic peritonitis.

Conclusions: Our results indicate that curcumin interferes with colonic inflammation partly through inhibition of the chemokine expression and through direct inhibition of neutrophil chemotaxis and chemokinesis.

Figure 1

ELISA for secreted MIP-2 (A) and Bio-Plex analysis for secreted KC (B), IL-1β (C) and MIP-1α (D) in the cell culture medium of peritoneal macrophages or YAMC colonocytes pretreated with or without curcumin (50μM) for 30 minutes at 37°C LPS prior to 2 hour (10 ng/mL; macrophages) of 4 hour (100 ng/mL, YAMC) treatment E. coli. LPS. *Statistically significant differences (p≤0.05) between values from cells treated with LPS alone and other respective treatments (ANOVA followed by Fisher PLSD post-hoc test).

Figure 2

(A) Flow cytometry analysis of PMN after magnetic selection. (B) Cytotoxity assay with PMN treated with and without curcumin at different concentrations (5 to 100μM) for 2 hours evaluated by adenylate kinase release (RLU, relative light units). (C) Trypan blue dye exclusion assay with PMN treated with and without curcumin at different concentrations (5 to 100μM) for 2 hours.

Figure 3

Chemotaxis Assay with calcein red-orange AM stained bone marrow-derived mouse neutrophils against conditioned medium obtained from intraperitoneal macrophages (A) or YAMC cells (B) treated with DMSO (control), 50 μM curcumin, LPS (10 ng/mL), or LPS with curcumin (the same treatment conditions as described in Fig. 1). *Statistically significant differences (p≤0.05) between values from cells treated with curcumin or curcumin/LPS and control or LPS treatment; # statistically significant differences between values from cells treated with LPS alone and other respective treatments (ANOVA followed by Fisher PLSD post-hoc test).

Figure 4

(A) Chemotaxis of untreated, calcein-stained neutrophils against recombinant MIP-2 (5–20 ng/mL) without or with 25 or 50 μM curcumin in the lower chamber of the ChemoTx® Chemotaxis System. * p≤0.05 MIP-2 vs. control; # p≤0.05 curcumin vs. control or curcumin/MIP-2 vs. MIP-2 alone. (B) Chemotaxis of neutrophils pretreated with 10–50μM of curcumin (30 min prior to the assay) against recombinant MIP-2 (20ng/mL) * p≤0.05 MIP-2 vs. control; # p≤0.05 Curcumin 10μM/MIP-2 vs. MIP-2 alone; † Curcumin 25 or 50μM vs. Curcumin 10μM/MIP-2 or MIP-2 alone. (C) Chemokinesis assay of PMN with recombinant MIP-2 (20ng/mL) placed in the top and bottom chamber. PMNs were pretreated with curcumin (10, 25 or 50μM) 30 minutes prior to the assay. * p≤0.05 10–25μM curcumin vs. DMSO; # p≤0.05 50 μM curcumin vs. control or 10–20 μM curcumin (ANOVA followed by Fisher PLSD post-hoc test). (D) Chemotaxis of untreated, calcein-stained neutrophils against recombinant KC (75 ng/mL) without or with 25 or 50 μM curcumin in the lower chamber of the ChemoTx® Chemotaxis System. * p≤0.05 KC vs. control; # p≤0.05 KC/curcumin vs. KC; ** control vs. curcumin alone.

Figure 5

(A) Chemotaxis of untreated, calcein-stained human- neutrophils against recombinant IL-8 (20 ng/mL) without or with 25 or 50 μM curcumin in the lower chamber of the ChemoTx® Chemotaxis System. * p≤0.05 IL-8 vs. control; # p≤0.05 IL-8/curcumin vs. control or curcumin/IL-8 vs. IL-8 alone. (B) Chemotaxis of neutrophils pretreated with 50μM of curcumin (30 min prior to the assay) against recombinant IL-8 (20ng/mL) * p≤0.05 IL-8 vs. control; # p≤0.05 Curcumin 50μM/IL-8 vs. IL-8 alone or IL-8/curcumin vs. control. (C) Chemokinesis assay of PMN with recombinant IL-8 (20ng/mL) placed in the top and bottom chamber. PMNs were pretreated with curcumin (25 or 50μM) 30 minutes prior to the assay. * p≤0.05 10–20μM curcumin vs. DMSO; # p≤0.05 50 μM curcumin vs. control or 10–25 μM curcumin (ANOVA followed by Fisher PLSD post-hoc test)

Figure 6

Under agarose neutrophil migration assay: F-actin was labeled after migration with Alexa-647 conjugated phalloidin and nuclei were counterstained with sytox green and cells were imaged under a confocal laser scanning microscope. (A) Control DMSO-treated PMN or PMN pretreated with 10–50 μM of curcumin for 30 min prior to the assay were loaded into different wells cut in 1.6% agarose gel. Recombinant MIP-2 (20ng/ml) was placed in the central well. Each well was placed at equal distance from each other. High magnification images from PMN treated with DMSO or 25 μM curcumin are depicted as crop-outs. (B) Curcumin was homogeneously added to the gel at 25 or 50 μM. PMN or recombinant MIP-2 (20ng/ml) were loaded into adjacent, evenly spaced wells.

Figure 6

Under agarose neutrophil migration assay: F-actin was labeled after migration with Alexa-647 conjugated phalloidin and nuclei were counterstained with sytox green and cells were imaged under a confocal laser scanning microscope. (A) Control DMSO-treated PMN or PMN pretreated with 10–50 μM of curcumin for 30 min prior to the assay were loaded into different wells cut in 1.6% agarose gel. Recombinant MIP-2 (20ng/ml) was placed in the central well. Each well was placed at equal distance from each other. High magnification images from PMN treated with DMSO or 25 μM curcumin are depicted as crop-outs. (B) Curcumin was homogeneously added to the gel at 25 or 50 μM. PMN or recombinant MIP-2 (20ng/ml) were loaded into adjacent, evenly spaced wells.

Figure 7

(A) ELISA for PIP3 concentration (pMol/10⁶ cells) in bone marrow derived-neutrophil treated with 10–50μM of curcumin 5 minutes prior to a 30-second stimulation with MIP-2 (20ng/ml). (B) Western blot analysis of phospho-AKT (Ser⁴⁷³) or to phospho-p44/42 MAPK (Thr²⁰²/Tyr²⁰⁴) in PMNs treated with 10–50μM of curcumin 5 minutes prior to a 30-second stimulation with MIP-2 (20ng/ml). Total AKT and total ERK were evaluated as loading control. * p≤0.05 curcumin/MIP-2 vs. control or MIP-2 alone (ANOVA followed by Fisher PLSD post-hoc test). (C) Densitometric analysis of AKT and (D) ERK phosphorylation in response to MIP-2 with or without 10–50μM curcumin.

Figure 8

Effect of curcumin on neutrophil recruitment in thioglycollate induced aseptic peritonitis. (A) BALB/c mice received three i.p. injections of curcumin or vehicle alone every 12 hours in a total volume of 80μl. 4 hours after the last i.p. injection of curcumin or vehicle, peritonitis was induced by i.p. injection of 500μl of 3% thioglycollate (wt/vol; Sigma). 3 hours later, intra-peritoneal cells lavaged with 5mL of cold RPMI 1640 and the number of cells was determined using the Vi-Cell XR automatic cell counter and viability analyzer (Beckman-Coulter). (B) Giemsa staining of the lavaged peritoneal cells in PBS or thioglycolate injected mice. (C) The effect of curcumin on peritoneal neutrophil recovery in aseptic peritonitis. * p≤0.05 curcumin/thioglycollate vs. all other treatments (ANOVA followed by Fisher PLSD post-hoc test).