Macrophage-Immunomodulatory Actions of Bovine Whey Protein Isolate, Glycomacropeptide, and Their In Vitro and In Vivo Digests

Abstract

Whey protein isolate (WPI) consists of an array of proteins and peptides obtained as a byproduct of the cheesemaking process. Research suggests that WPI, along with its peptides such as glycomacropeptide (GMP), possesses immunomodulatory properties. These properties hold potential for alleviating the adverse effects of inflammatory conditions such as inflammatory bowel disease. Although promising, the immunoregulatory properties of the digested forms of WPI and GMP-those most likely to interact with the gut immune system-remain under-investigated. To address this knowledge gap, the current study examined the effects of in vitro-digested WPI and GMP, in vivo-digested WPI, and undigested WPI and GMP on the secretion of pro-inflammatory cytokines (TNF-α and IL-1β) in lipopolysaccharide-stimulated macrophage-like cells. Our results indicate that digested WPI and GMP reduced the expression of TNF-α and IL-1β, two pro-inflammatory cytokines. Whole WPI had no effect on TNF-α but reduced IL-1β levels. In contrast, in vivo-digested WPI reduced TNF-α but increased IL-1β. Undigested GMP, on the other hand, increased the secretion of both cytokines. These results demonstrate that digestion greatly modifies the effects of WPI and GMP on macrophages and suggest that digested WPI and GMP could help mitigate gastrointestinal inflammation. Further clinical studies are necessary to determine the biological relevance of WPI and GMP digestion products within the gut and their capacity to influence gut inflammation.

Keywords: Crohn’s disease; caseinmacropeptide; caseinomacropeptide; immunity; milk; ulcerative colitis.

Figure 1

Percentage change in TNF-α concentration for each whey protein isolate sample type (whey protein isolate (WPI), in vitro-digested WPI (WPID), and intestinally digested WPI (WPIINT) at each concentration level) compared to the LPS-only control. Each treatment was administered at 10 (blue), 100 (red), and 1000 (green) µg/mL in medium. Bar heights represent mean values, with brackets representing ± standard deviations (n = 6 technical replicates (3 days of cell experiment replication, duplicate wells on each day)). Stars above each bar indicate statistically significant differences from the LPS-only control based on the two-sided t-test (p < 0.05). Letters indicate the significance of differences in percent change of TNF-⍺ from the LPS-only control among WPI, WPID, and WPIINT at each concentration based on a multi-factor ANOVA followed by Tukey’s HSD tests.

Figure 2

Percentage change in IL-1β concentration for each whey protein isolate sample type (whey protein isolate (WPI), in vitro-digested WPI (WPID), and intestinally digested WPI (WPIINT) at each concentration level) compared with the LPS-only control. Each treatment was administered at 10 (blue), 100 (red), and 1000 (green) µg/mL in medium. Bar heights represent mean values, with brackets representing ± standard deviations (n = 6 technical replicates (3 days of cell experiment replication, duplicate wells on each day)). Stars above each bar indicate statistically significant differences from the LPS-only control based on the two-sided t-test (p < 0.05). Letters indicate the significance of differences in percentage change of IL-1β from the LPS-only control among WPI, WPID, and WPIINT at each concentration based on a multi-factor ANOVA followed by Tukey’s HSD tests.

Figure 3

Percentage change in TNF-α concentration for each glycomacropeptide sample type (Glycomacropeptide (GMP), in vitro-digested GMP (GMPD), at each concentration) compared to the LPS-only control. Each treatment was administered at 10 (blue), 100 (red), and 1000 (green) µg/mL in medium. Bar heights represent mean values, with brackets representing ± standard deviations (n = 6 technical replicates (3 days of cell experiment replication, duplicate wells on each day)). Stars above each bar indicate statistically significant differences from the LPS-only control based on the two-sided t-test (p < 0.05). Letters indicate the significance of differences in percentage change of TNF-⍺ from the LPS-only control among GMP and GMPD at each concentration based on a multi-factor ANOVA followed by Tukey’s HSD tests.

Figure 4

Percentage change in IL-1β concentration for each glycomacropeptide sample type (glycomacropeptide [GMP], in vitro-digested GMP [GMPD], at each concentration) compared to the LPS-only control. Each treatment was administered at 10 (blue), 100 (red), and 1000 (green) µg/mL in medium. Bar heights represent mean values, with brackets representing ± standard deviations (n = 6 technical replicates (3 days of cell experiment replication, duplicate wells on each day)). Stars above each bar indicate statistically significant differences from the LPS-only control based on the two-sided t-test (p < 0.05). Letters indicate the significance of differences in percentage change of IL-1β from the LPS-only control among GMP and GMPD at each concentration based on a multi-factor ANOVA followed by Tukey’s HSD tests.