Long-Term Oral Administration of Hyperimmune Egg-Based IgY-Rich Formulations Induces Mucosal Immune Response and Systemic Increases of Cytokines Involved in Th2- and Th17-Type Immune Responses in C57BL/6 Mice

Abstract

Three hyperimmune egg-based formulations rich in immunoglobulin Y (IgY) were orally administered (daily, for up to 90 days) to C57BL/6 mice that were not microbially challenged. The serum levels of 32 cytokines were quantified every 30 days. Histopathology, hematology, and serum biochemistry investigations were also performed. As a sign of increased immune activity, lymphohistiocytic infiltrates were detected in the digestive tract and the liver after 30, 60, and 90 days of treatment. These infiltrates were also present in the lungs after 30 and 60 days, but not at 90 days. Blood analysis indicated systemic inflammation after 30 days of treatment: increases in pro-inflammatory cytokines, glycemia, total serum proteins, ALT, and ALP. After 60 and 90 days of treatment, the analyzed blood parameters showed mixed signs of both increased and decreased inflammation. The increased cytokines, which varied with formulation and time of exposure, indicated a combination of mostly Th17- and Th2-type immune responses. As the mice were healthy and housed in standardized sanitary conditions, and were not microbially challenged, the data were consistent with an interaction of IgY with the gut-associated lymphoid tissue as the main mechanism of action. This interaction generated a local immune response, which subsequently induced a systemic response.

Keywords: C57BL/6 mice; biochemistry; cytokines; hematology; histopathology; hyperimmune egg; immune response; immunoglobulin Y; interleukins; oral administration.

Figure 1

Bodyweight (mean ± SD) in the five experimental groups at the beginning of the experiment (T0) and at the three harvesting time points of 30 days (T30), 60 days (T60), 90 days (T90). The five groups received distilled water (DW), specific pathogen-free (SPF) egg, fresh hyperimmune egg (HE), freeze-dried hyperimmune egg (fdHE), or an IgY extract (Yext).

Figure 2

Histopathology aspects: (a) T30 Liver—lymphohistiocytic infiltrate; (b) T90 Liver—lymphohistiocytic infiltrate; (c) T30 Duodenum—lymphohistiocytic infiltrate; (d) T60 Duodenum—lymphohistiocytic infiltrate with eosinophils; (e) T90 Duodenum—lymphohistiocytic infiltrate with amyloidosis; (f) T90 Colon—lymphohistiocytic infiltrate with amyloidosis; (g) T90 Lung—without lymphohistiocytic infiltrate; (h) T30 Spleen—congestion caused by anesthesia and formaldehyde infusion; (i) T90 Kidney—without lymphohistiocytic infiltrate.

Figure 3

Serum levels (mean ± SD) of the cytokines that were increased in the treatment groups (HE, fdHE, Yext) compared with both control groups (DW, SPF): at the beginning of the experiment (T0), after 30 days (T30), 60 days (T60), and 90 days of treatment (T90).

Figure 4

The levels of serum biochemistry parameters (mean ± SD): alkaline phosphatase (ALP), alanine aminotransferase (ALT), glucose (GLU), total proteins (T-PRO), blood urea nitrogen (BUN), and creatinine (CREAT): at the beginning of the experiment (T0) and at the three harvesting time points of 30 days (T30), 60 days (T60), 90 days (T90).

Figure 5

Total WBC levels (mean ± SD): at the beginning of the experiment (T0) and at the three harvesting time points of 30 days (T30), 60 days (T60), 90 days (T90).

Figure 6

The levels of neutrophils (NEU), monocytes (MONO), and lymphocytes (LYM) in absolute numbers (10⁹/L) and percentages of total white blood cells (mean ± SD) at the beginning of the experiment (T0) and at the three harvesting time points of 30 days (T30), 60 days (T60), 90 days (T90).

Figure 7

Schematic representation of the experimental design.