Extracts of Feijoa Inhibit Toll-Like Receptor 2 Signaling and Activate Autophagy Implicating a Role in Dietary Control of IBD

Abstract

Background: Inflammatory bowel disease (IBD) is a heterogeneous chronic inflammatory disease affecting the gut with limited treatment success for its sufferers. This suggests the need for better understanding of the different subtypes of the disease as well as nutritional interventions to compliment current treatments. In this study we assess the ability of a hydrophilic feijoa fraction (F3) to modulate autophagy a process known to regulate inflammation, via TLR2 using IBD cell lines.

Method: Mouse embryonic fibroblasts (MEF) deleted for ATG5, and two intestinal epithelial cells HCT15 and HCT116, were used to test the anti-inflammatory effect of F3 after stimulating the cells with a TLR2 specific ligand PAM3CSK4.

Results: F3 was able to reduce TLR2 specific inflammation and stimulate autophagy in MEFs and HCT15 cells but not in HCT116 cells. The anti-inflammatory effect was reduced in the MEF cells deleted for ATG5. In addition, the activation of autophagy by F3 was enhanced by PAM3CSK4.

Conclusion: F3 of feijoa can interact with cells via a TLR2 specific mechanism and reduce Nuclear factor kappa B (NF-κB) activation in part due to stimulation of autophagy. These results suggest that there is potential benefit in using feijoa extracts as part of dietary interventions to manage IBD in patients.

Fig 1. Feijoa F3 activates autophagy in MEF cells.

Cells were treated with feijoa F3 at 10mg/ml, Torin1 at 1μM/ml, with and without 100ng/ml PAM3CSK4 stimulation. A. Shows the LC3B (marker of autophagy) staining. The original images show puncta in cells that represent autophagosomes. The rendered images are created using the Imaris spot function where the spots in the cells are spheres pseudo colored and represent the autophagosomes. The MEF Atg 5^-/- showed no puncta B. The puncta were quantified using the spot function in Imaris. Each experimental condition for solvent, media, and feijoa F3 had 3 biological replicates with two technical replicates (giving a maximum of 6 data points) and Torin1 had 2 biological replicates with two technical replicates (giving a maximum of 4 data points). Horizontal line represents median.

Fig 2. Feijoa F3 inhibits NF-κB activity in A. MEF cells and B. MEF Atg5^-/- cells.

Cells were treated with feijoa F3 at 10mg/ml, Torin1 at 1μM/ml, with and without 100ng/ml PAM3CSK4 stimulation. A and B show the normalized percentage NF-κB activation. Percentage inflammation was calculated using Eq 3. A percentage of greater than 100 shows increased inflammatory response in the presence of the ligand when compared to media (no treatment); and a percentage of less than 100 suggests a decrease in the inflammatory response in the presence of the ligand when compared to media (no treatment) meaning that a condition has inhibited NF-κB signaling. Error bars represent standard deviation (n = 6). ** P-value <0.001 * <0.05.

Fig 3. Feijoa F3 activates autophagy and inhibits NF-κB activity in HCT15 cells.

Cells were treated with media, Torin1 (1μM/ml), solvent and feijoa F3 (10mg/ml), in the presence and absence of 100ng/ml PAM3CSK4 stimulation. A and B show the number of autophagosomes in each cell. The puncta were quantified using the spot function in Imaris. Each experimental condition for solvent, media, and feijoa F3 had 3 biological replicates with two technical replicates (giving a maximum of 6 data points) and Torin1 had 2 biological replicates with two technical replicates (giving a maximum of 4 data points). Horizontal line represents median. B is an image representation of A. The spots in the cells are the spheres pseudo colored by the Imaris spot function representing the autophagosome puncta that were stained for by LC3B. C. shows western blot analysis of autophagy. The Western blot show LC3B I (16kDa) and II (18kDa) protein bands. β-actin (42 kDa) was used as a control. The bottom graph is the semi quantification of the LC3 I and II band intensity scores, adjusted against β-actin band intensity scores. D shows the normalized percentage NF-κB activation. Percentage inflammation was calculated using Eq 3. A percentage of greater than 100 shows increased inflammatory response in the presence of the ligand when compared to media (no treatment); and a percentage of less than one suggests a decrease in the inflammatory response in the presence of the ligand when compared to media (no treatment) meaning that a condition has inhibited NF-κB signaling. Error bars represent standard deviation of (n = 6). * P-value <0.05.

Fig 4. Feijoa F3 does not activate autophagy or inhibit NF-κB activity in autophagy defective HCT116 cells.

Cells were treated with media, Torin1 (1μM/ml), solvent and feijoa F3 (10mg/ml), in the presence and absence of 100ng/ml PAM3CSK4 stimulation. A and B show the number of autophagosomes in each cell. The puncta were quantified using the spot function in Imaris. Each experimental condition for solvent, media, and feijoa F3 had 3 biological replicates with two technical replicates (giving a maximum of 6 data points) and Torin1 had 2 biological replicates with two technical replicates (giving a maximum of 4 data points). Horizontal line represents median. B is an image representation of A. The spots in the cells are the spheres pseudo colored by the Imaris spot function representing the autophagosome puncta that were stained for by LC3B. C. shows western blot analysis of autophagy. The westerns show LC3B I (16kDa) and II (18kDa) protein bands. β-actin (42 kDa) was used as a control. The bottom graph is the semi quantification of the LC3 I and II band intensity scores, adjusted against β-actin band intensity scores. D shows the normalized percentage NF-κB activation. Percentage inflammation was calculated using Eq 3. A percentage of greater than 100 shows increased inflammatory response in the presence of the ligand when compared to media (no treatment); and a percentage of less than 100 suggests a decrease in the inflammatory response in the presence of the ligand when compared to media (no treatment) meaning that a condition has inhibited NF-κB signaling. Error bars represent standard deviation of (n = 6).