Toxicological Characteristics of Bacterial Nanocellulose in an In Vivo Experiment-Part 2: Immunological Endpoints, Influence on the Intestinal Barrier and Microbiome

Abstract

Bacterial nanocellulose (BNC) is considered a promising alternative to microcrystalline cellulose, as well as an ingredient in low-calorie dietary products. However, the risks of BNC when consumed with food are not well characterized. The aim of this study is to investigate the impact of BNC on immune function, the intestinal microbiome, intestinal barrier integrity, and allergic sensitization in subacute experiments on rats. Male Wistar rats received BNC with a diet for eight weeks in a dose range of 1-100 mg/kg of body weight. The measurements of serum levels of cytokines, adipokines, iFABP2, indicators of cellular immunity, composition of the intestinal microbiome, and a histological study of the ileal mucosa were performed. In a separate four-week experiment on a model of systemic anaphylaxis to food antigen, BNC at a dose of 100 mg/kg of body weight did not increase the severity of the reaction or change the response of IgG antibodies. Based on dose-response effects on immune function, the non-observed adverse effect level for BNC was less than 100 mg/kg of body weight per day. The effects of BNC on the gut microbiome and the intestinal mucosal barrier were not dose-dependent. Data on the possible presence of prebiotic effects in BNC have been obtained.

Keywords: anaphylaxis; bacterial cellulose; cytokines; intestinal barrier; intestinal microbiome; lymphocytes; nanofibers; rats; toxicity.

Figure 1

Immunological parameters of rats in an 8-week toxicological experiment. (a) Total leukocyte count in peripheral blood, 10⁹*L⁻¹; (b) total lymphocyte count, 10⁹*L⁻¹; (c) total number of neutrophils, 10⁹*L⁻¹; (d) the number of CD4+ cells in % of the total number of CD3+ cells; (e) immunoregulatory index (the ratio of CD4+ to CD8+ cells); (f) serum IL-17A content, pg/ml; and (g) serum leptin content, pg/mL. Number of animals 10 (a–e) and 8 (f,g) per group. * The difference is significant for groups connected by an arrow, p < 0.05, Mann–Whitney test.

Figure 2

Indicators of the microbiome of the cecum of rats in an 8-week toxicological experiment. Content: (a) aerobic microorganisms; (b) hemolytic aerobes; (c) lactobacilli; (d) enterococci; and (e) molds. (f) A diagram (heat map) of the degree of discrepancy between the microbiome of the animals of the experimental groups and the control following the direction and statistical significance of the changes detected. The number of animals: 8 per group. Boxplot charts show the median value (horizontal bar), quartile range (box), range of change (bars), and outliers (circles). *—see Figure 1.

Figure 3

Indicators of the morphofunctional state of the ileum mucosa of rats in an 8-week toxicological experiment. Representative micrographs of the intestinal wall of rats from group 2 (a,b) and group 3 (c) in relation (arrows, dotted lines) to individual serum levels of fatty acid binding protein (iFABP2) (d); the proportion in % of the villi with a disturbed epithelial layer (e); average length of villi and depth of crypts by groups of animals, µm (f); and the ratio of the length of the villi to the depth of the crypts (g). Number of animals per group: 10 (d); 4 (e–g) with at least 45 pairs of villi and crypts examined on all slides from each group. *—a significant difference from group 1 (control), p < 0.05, Mann–Whitney test. Hematoxylin-eosin staining, ×100 magnification (a); ×50 (b,c).

Figure 4

The results of reproducing the reaction of systemic anaphylaxis to OVA in rats of the control group and treated with BNC at a dose of 100 mg/kg b.w. (a) Distribution of animals of the two groups according to the severity of the anaphylaxis reaction, expressed in points. (b) Individual serum concentrations of specific IgG4 antibodies to OVA, mg/mL in two groups. The number of animals: 24 per group.