1. High levels of fish oil enhance neutrophil development and activation and influence colon mucus barrier function in a genetically susceptible mouse model 
David M. Duriancik, Sarah S. Comstock, Ingeborg M. Langohr, Jenifer I. Fenton 
Journal of Nutritional Biochemistry 
Volume 26, Issue 11, Pages (November 2015)
DOI: /j.jnutbio
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Fig. 1
FO increases neutrophil development in the spleen and bone marrow. A, Spleen neutrophils as a percentage (CON; n=10, 5 male/5 female, FO; n=30, 15 male/15 female, p value Welch’s corrected) and total cell number (CON; n=10, FO; n=30, p value Welch’s corrected). Spleen neutrophil percentage separated by sex comparing FO to control. B, Bone marrow neutrophils as a percentage and total cell number (CON; n=10, 5 male/5 female, FO; n=30, 15 male/15 female, p value Welch’s corrected). Bone marrow neutrophil percentage separated by sex comparing FO to control. C, CD11b expression on spleen neutrophils (CON; n=10, 5 male/5 female, FO; n=30, 15 male/15 female). Spleen neutrophil CD11b expression separated by sex comparing FO to control. There was no significant difference in any of these data between the different FO diets. Therefore all FO diets were combined.
Journal of Nutritional Biochemistry  , DOI: ( /j.jnutbio )
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Fig. 2
FO reduces neutrophil function ex vivo in female mice. A, Flow cytometric gating for neutrophil ROS production. Bone marrow cells were loaded with DCF and stimulated with PMA. Stimulation of bone marrow cells with PMA did not alter the forward scatter and side scatter characteristics of the cells used to gate live cells and neutrophils were gated as Gr-1hi. To account for the increase in CD11b expression in cells stimulated with PMA compared to no target, neutrophils were gated as CD11b+ including both dim and high expression. Neutrophils were confirmed with high side scatter properties and MFI of DCF was measured. B, Graphical representation of neutrophil ROS production (DCF median fluorescence intensity) separated by sex. Sample size of each group was 4–5 (CON male NT n=5, CON male PMA n=5, FO male NT n=5, FO male PMA n=5, CON female NT n=5, CON female PMA n=5, FO female NT n=5, FO female PMA n=4). FO-fed mice were fed Menhaden oil.
Journal of Nutritional Biochemistry  , DOI: ( /j.jnutbio )
Copyright © 2015 Elsevier Inc. Terms and Conditions

4. Fig. 3
FO results in colon inflammation and histopathology. A, Graphical representation of intestinal inflammation scores for sex combined (CON; n=10, 5 male/5 female, FO; n=30, 15 male/15 female, Welch’s corrected p value for analysis of sex combined and male data, female analysis was not Welch’s corrected) and separated by sex. B, Representative images of colon (400× images) and cecal (600× images) inflammation. Moderate numbers of granulocytes (*) infiltrate the lamina propria in the colon and cecum. Additionally, there are multiple granular leukocytes (black arrow) and occasional mitotic figures (red arrow) in the crypt epithelium. Possible bacteria were visible in rare crypts of this animal (circle). There was no significant difference in any of these data between the different FO diets. Therefore all FO diets were combined.
Journal of Nutritional Biochemistry  , DOI: ( /j.jnutbio )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Fig. 4
FO feeding increases neutrophil recruitment to the colon and cecum. A, Increased neutrophil percentage of live hematopoietic-derived cells in FO-fed mice (CON; n=19, 9 male/10 female, FO; n=30, 15 male/15 female Welch’s corrected p value for analysis of sex combined and male data, analysis of female data was not Welch’s corrected). B, CD11b expression on neutrophils (CON; n=5 male/9 females, FO; n=5 males/9 females). C, Eosinophil percentage of live hematopoietic-derived cells in colon and cecum (CON; n=9, 4 male/5 female, FO; n=20, 10 male/10 female). FO-fed mice were fed Menhaden oil.
Journal of Nutritional Biochemistry  , DOI: ( /j.jnutbio )
Copyright © 2015 Elsevier Inc. Terms and Conditions

6. Fig. 5
FO reduces mucus layer and goblet cells. A, Representative images of colon and cecal mucus layer and goblet cells stained with alcian blue/PAS at 400×. Multiple goblet cells aggregate at the lower aspect of the cecal glands. The mucus layer is covered by an artifactually disrupted mucus layer (*). In FO-fed mice, fewer and frequently individualized goblet cells are in the cecal glands, accompanied by a thinner superficial mucus layer (arrow) than those seen in the control mice. Goblet cells are uniformly distributed throughout the colonic glandular epithelium. The thick, stratified, generally firmly attached inner mucus layer impervious to bacteria (i) is artifactually detached from the mucus layer, overlaid, as expected, by a second, outer mucus layer interspersed by numerous bacteria (o). Several colonic glands have reduced numbers of goblet cells. Additionally in this region of the colon, the normally bilayer mucus is replaced by a single layer of artifactually separated thin strands of mucin (*). B, Mucus layer thickness (Cecum and colon, CON; n=10, 5 male/5 female, FO; n=9, 5 male/4 female, Welch’s corrected p value for cecum only) and goblet cell number (Cecum and colon, CON; n=10, 5 male/5 female, FO; n=9, 5 male/4 female) graphical summary. C, Fecal IgA quantitation (CON; n=10, 5 male/5 female, FO; n=30, 15 male/15 female, Welch’s corrected p value, no difference between different FO diets). D, FITC–dextran absorption (Male, CON; n=10, FO; n=9, Female, CON; n=10, FO; n=10, Welch’s corrected p value). FO-fed mice were fed Menhaden oil.
Journal of Nutritional Biochemistry  , DOI: ( /j.jnutbio )
Copyright © 2015 Elsevier Inc. Terms and Conditions

7. Fig. 6
Colonic B cells of FO-fed mice are less activated and germinal center-like. A, Colon and cecal B cell percentage of live hematopoietic-derived cells (CON; n=19, 9 male/10 female, FO; n=30, 15 male/15 female). B, Expression of CD19 on colon and cecal B cells (Cecum and colon, CON; n=19, 9 male/10 female, FO; n=30, 15 male/15 female, Welch’s corrected p value) and CD19 expression separated by sex (Welch’s corrected analysis was not used). C, Percentage of colon and cecal B cells expressing PNA (CON; n=6, 3 male/3 female, FO; n=15, 7 male/8 female), a marker of germinal center B cells and separated by sex. FO-fed mice were fed Menhaden oil.
Journal of Nutritional Biochemistry  , DOI: ( /j.jnutbio )
Copyright © 2015 Elsevier Inc. Terms and Conditions