Yolkin restores cellular immune response and content of T and B cells in lymphoid organs of cyclophosphamide-immunocompromised mice

Eight-week-old female BALB/c mice were purchased from Envigo (Limburg, the Netherlands). They were kept under 12 h/12 h light and dark cycles and given a commercial pelleted feed and filtered tap water ad libitum. The Local Ethics Committee for Animal Experiments of the Hirszfeld Institute of Immunology in Wrocław, Poland gave consent under No. 014/2023 for the experiments to be performed. Consideration to the principle of the 3Rs (replacement, reduction and refinement) was given in the experimental design.

Cyclophosphamide (Endoxan) was from Baxter Oncology (Halle, Germany). Hanks’ balanced salt solution (HBSS), phosphate buffered saline (PBS), bovine serum albumin (BSA), 2 M H₂SO₄, May–Grünwald–Giemsa stain, Histopaque 1077 1.077 g/mL, incomplete Freund’s adjuvant, oxazolone (OXA) and ovalbumin (OVA) were from Merck (Munich, Germany). Rat anti-mouse CD19⁺ fluorescein isothiocyanate (FITC)/CD3⁺:R-phycoerythin (RPE) (cat. No. DC035), rat anti-mouse CD4⁺:FITC/CD8⁺:RPE (cat. No. DC034), rat anti-mouse CD169:FITC (cat. No. MCA884F) and hamster antimouse CD11c:Alexa488 (cat. No. MCA1369A488) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Rat anti-mouse CD14:RPE (cat. No. AB-465563) was a product of Invitrogen (Waltham, MA, USA), goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody (cat. No. 1030-05) was supplied by SouthernBiotech (Birmingham, AL, USA) and mouse regulatory T cell (Treg) staining kit #1 is in the eBioscience range (Carlsbad, CA, USA). As a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB – cat No. 00-4201-56) was used from Bender MedSystems/Thermo Fisher Scientific (Vienna, Austria). Alum adjuvant (Alhydrogel adjuvant 2%) was from InvivoGen (San Diego, CA, USA). Yolkin was isolated from hen eggs and characterised by polyacrylamide gel electrophoresis as previously described (23). The preparation contained two predominant proteins of 40 and 35 kDa with traces of proteins with 30 kDa molecular weight.

The experiment was carried out in two models: contact sensitivity (CS) to OXA and humoral response to OVA. In the model of CS to OXA the mice in the experiment were divided into four groups (in each of the group n = 16). The negative control group were injected intraperitoneally (i.p.) with 0.9% NaCl solution in a 0.1 mL volume instead of CP, and drank tap water. The yolkin-only group were likewise injected i.p. with 0.9% NaCl solution instead of CP, and drank tap water with yolkin added to it at 5 μg/mL between days 3 and 15. The CP-only group received CP i.p. in two injections on days 1 and 2 at 50 mg/kg b.w. in a 0.1 mL volume (overall dose 100 mg/kg b.w.), and drank tap water. The CP- and-yolkin group likewise received CP and drank tap water with the same concentration of yolkin for the same period as the yolkin-only group. On day 16 half of each experimental group (n = 8) were anaesthetised and sacrificed by cervical dislocation, and the lymphoid organs were exsected for phenotypic determinations of the immune cells. The rest of each experimental group (n = 8) were immunised with OXA on day 16, the CS reaction was elicited on day 21, and the next day (after 24 h) the ear oedema was measured. The experimental approach is presented in Fig. 1A.

Fig. 1.

Experimental design. Immunological determinations in (A) the model of contact sensitivity to oxazolone (OXA) and (B) the model of humoral response to ovalbumin (OVA). CP – cyclophosphamide

In the model of the humoral response to OVA the mice in the experiment were divided into four groups (in each of the group n = 16). The negative control group were injected i.p. with 0.9% NaCl solution in a 0.1 mL volume instead of CP, and drank tap water. The yolkin-only group were likewise injected i.p. with 0.9% NaCl solution instead of CP, and drank tap water with yolkin added to it at 5 μg/mL between days 3 and 29. The CP-only group received CP i.p. in two injections on days 1 and 2 at 50 mg/kg b.w. in a 0.1 mL volume (overall dose 100 mg/kg b.w.), and drank tap water. The CP-and-yolkin group likewise received CP and drank tap water with the same concentration of yolkin for the same period as the yolkin-only group. On day 30 half of each experimental group (n = 8) were anaesthetised, bled and sacrificed by cervical dislocation, and the lymphoid organs were isolated for phenotypic determinations of the immune cells. The rest of each experimental group (n = 8) were sensitised with OVA on day 30 and after another 21 d (day 51 of the experiment) were anaesthetised, bled and sacrificed by cervical dislocation. The sera were isolated and kept frozen at –80° C until determination of level of anti-OVA IgG antibodies. The experimental approach is presented in Fig. 1B.

The test was performed according to Noonan and Halliday, with some minor modifications (21). Mice were shaved on the abdomen and 100 μL of 0.5% OXA (the sensitising dose of antigen), dissolved in acetone, was applied to the skin. After 5 d, 50 μL of 1% OXA (the eliciting dose of antigen) was applied to both sides of the auricles. The ear thickness was measured 24 h later by means of spring callipers with an accuracy of 0.05 mm. The results were determined as antigen-specific increases of the ear thickness, i.e. baseline measurements were subtracted (these having been measured in the mice given only the eliciting dose of antigen; an additional mouse group/n = 8/was used).

Ovalbumin was initially dissolved in 0.9% NaCl (0.2 mg/mL) and mixed with the alum adjuvant at 1:1 v/v (50 μL of OVA plus 50 μL of the adjuvant). Mice were immunised subcutaneously in the neck, with 10 μg of OVA (100 μL/mouse) emulsified with the adjuvant at 2%.

A 96-well plate (Nunc MaxiSorb; ThermoScientific, Roskilde, Denmark) was coated overnight at 4°C with 1 μg/mL OVA in PBS. The plate was then blocked for 2 h at room temperature with a buffer (1% BSA and 0.01% Tween-20 in PBS). A volume of 100 μL of tested sera, diluted 1:500 and 1: 1250 in assay diluent (0.1% BSA and 0.01% Tween-20 in PBS) was added and the plate was incubated for 2 h at room temperature. Each serum sample was tested in duplicate. A series of ten serial dilutions of a positive reference serum was also added to each plate, and each dilution was also tested in duplicate. This positive reference serum was prepared by pooling aliquots from six of the obtained control serum samples that had high antibody titres. The reference serum was arbitrarily defined as containing 1 × 10³ units of antibody activity per millilitre. The lowest dilution of the positive reference serum used in the test was 1:1000. Goat anti-mouse IgG HRP-conjugated antibody was added to the wells and incubated at room temperature for 1 h. Between each step and the next, the plate was washed four times (five times after the addition of the HRP-conjugated antibody) using a wash buffer (PBS containing 0.05% Tween 20). A 100 μL volume of TMB substrate solution was added to each well and the plates were incubated at room temperature for 10 min. The reaction was stopped by adding 100 μL of 2M H₂SO₄, and absorbance was measured at 450/570 nm using a microplate reader. A reference curve was constructed by plotting the absorbance of each reference sample against its arbitrary antibody units per mL, modelled using a four-parameter logistic fit of the data (Assayfit Pro; AssayCloud, Nijmegen, the Netherlands). Sample antibody units were read off this curve and reported as antibody units/mL.

The phenotypic determinations of cells were described in detail in a previous article (23). Briefly, lymphatic organs, including thymuses, spleens, marrow from femurs, and mesenteric lymph nodes, were exsected from euthanised mice. The marrow cavities of the femurs were washed with HBSS to obtain bone marrow cells, while other organs were minced and disaggregated through a nylon mesh into PBS. Splenocytes, thymocytes and mesenteric lymph node cells were centrifuged on a layer of Histopaque 1077 to obtain lymphocytes from the interphase. A trypan blue dye-exclusion assay revealed 90–98% viability of cell suspensions. The lymphocytes from the thymus, spleen and mesenteric lymph nodes (at a concentration of 1 × 10⁷ cells/mL) were stained with CD4⁺:FITC/CD8⁺:RPE dual-colour reagent. The splenocytes, bone marrow cells and lymphocytes from mesenteric lymph nodes were also stained with CD19⁺:FITC/CD3⁺:RPE dual-colour reagent, and splenocytes were additionally stained with rat anti-mouse CD14:RPE and rat anti-mouse CD169:FITC monoclonal antibodies. Hamster anti-mouse CD11c:Alexa488 antibody was also used to stain bone marrow cells. All antibodies were used at the dilutions recommended by the manufacturer. Cells were analysed using flow cytometry.

Percentages of Tregs in splenocytes were evaluated using the commercial mouse regulatory T cell staining kit # 1 following the manufacturer’s instructions. Cells were analysed using flow cytometry, identifying Tregs based on CD25 and Foxp3 expression levels within the CD4⁺ lymphocyte subset (37).

A Brown–Forsythe test was used to determine the homogeneity of variance between groups. When the variance was homogenous, a one-way analysis of variance was applied, followed by post-hoc comparisons with Bonferroni or Tukey’s tests to evaluate the significance of the difference between groups. Nonparametric data were evaluated with Kruskal–Wallis analysis of variance. The results are presented as mean values ± standard error of the mean. Significance was determined at P-value < 0.05. The data were analysed statistically using Statistica for Windows software (version 13.3; Dell, Round Rock, TX, USA).

Changes in body weight of the experimental mice, presented as percentages of initial body weight, are shown in Fig. 2. The measurements revealed about a 5% loss of body weight in CP-treated mice 3–9 d following CP administration. The mice that were additionally administered yolkin in their drinking water lost less body weight, particularly during the period of 13–15-d post treatment.

Fig. 2.

Changes in cyclophosphamide (CP)-immunocompromised mouse body weight during the experimental period. The results are presented as % of initial body weight (mean values) of mice from individual experimental groups from both research models: contact sensitivity to oxazolone and humoral response to ovalbumin. Control – control mice given only 0.9% NaCl; Yolkin – mice given 0.9% NaCl and yolkin; CP/Control – mice given only CP; CP/Yolkin – mice given CP and yolkin

The treatment of mice with CP significantly inhibited the effectual phase of CS, as measured by the auricle thickness (Fig. 3). Treatment with yolkin alone led to a non-significant decrease in this parameter. The application of yolkin to drinking water for 13 d after administration of CP restored the CS response to 85.5% of the control values.

Fig. 3.

Antigen specific increase in ear oedema in cyclophosphamide (CP)-immunocompromised mice expressing contact sensitivity to oxazolone. The antigen-specific increase of ear thickness (the difference obtained by subtracting the baseline measurement from the measurement taken from sensitised mice) is presented as the mean value of ear thickness measured in eight mice (16 determinations) in mm ± standard error of the mean. * – statistically significant (P-value < 0.05); Control – control mice given only 0.9% NaCl; Yolkin – mice given 0.9% NaCl and yolkin; CP/Control – mice given only CP; CP/Yolkin – mice given CP and yolkin

Table 1 summarises the changes in the total number of cells, cell types and cell subpopulations in the lymphoid organs of mice subjected to CP treatment and exposure to yolkin for 13 d in drinking water.

The total cell numbers in the investigated organs decreased in all organs, but statistically significant decreases occurred only in the lymph nodes and bone marrow. The cell loss in these organs was not reversed in mice ingesting yolkin. The decreases in respective T cell subpopulations also took place in the thymus, although a significant decrease occurred only in the CD4^–CD8^– subset, the level of which was not changed by yolkin. The losses of T cell subsets and CD19⁺ B cells were quite marked in the spleen after CP treatment. Although not statistically significant, they were associated with a reversal of CD3⁺, CD4⁺ and CD8⁺ T cell loss to respective 88%, 89% and 76.6% of the values observed in the untreated control group. These changes did not extend to the levels of macrophages in the spleen. A decline of cell numbers was also evident in the mesenteric lymph nodes, and B cells were particularly affected. No reconstitution of the cell numbers by yolkin was observed. In addition, no changes in the B cell numbers in the bone marrow nor in T regulatory CD25⁺Foxp3⁺ cells in the spleen were found.

The results show that the antibody levels were enhanced to some degree by the treatment with yolkin on its own and slightly diminished by CP treatment (Fig. 4). The treatment of CP-immunocompromised mice with yolkin showed only a tendency to not significantly elevate the serum antibody concentration above the value noted in the untreated control mice. In general, the registered changes in the antibody levels were minor.

Fig. 4.

The levels of antibodies to ovalbumin in cyclophosphamide (CP)-immunocompromised mice followed by 26-d oral treatment with yolkin. The data are presented as mean and individual values of the antibody levels on a logarithmic scale. Control – control mice given only 0.9% NaCl; Yolkin – mice given 0.9% NaCl and yolkin; CP/Control – mice given only CP; CP/Yolkin – mice given CP and yolkin

The total cell numbers in the thymus, expressed as percentages, significantly decreased after treatment of mice with CP (Table 2). Although yolkin did not increase the cell numbers in a significant manner after CP treatment, numbers did not significantly differ from those observed in untreated control mice. The total number of splenocytes significantly decreased (by 41.9%) after CP treatment and did not recover after administration of yolkin. However, in the case of mesenteric lymph nodes, a significant decrease in the cell content was partially restored by yolkin and these cell numbers were not significantly different from the values registered in the untreated control mice. Notably, the most pronounced changes with regard to the effect of CP and yolkin were noted in the bone marrow, where a very significant depletion of cell numbers mediated by CP was completely restored by the yolkin treatment.

The analysis of changes in cell numbers of respective cell phenotypes in the lymphoid organs showed that the CD4⁺CD8⁺ cell subset of thymocytes was significantly reduced and partially recovered following yolkin treatment. In the spleen, there were significant decreases in the T cells of the CD3⁺, CD8⁺ and CD4⁺ phenotypes after CP treatment and CD19⁺ B cells. In the case of CD8⁺ cells, yolkin significantly restored the cell level registered in CP-treated mice. Of interest is that yolkin alone lowered CD8⁺ T cell number. In addition, the number of splenic regulatory T cells (CD25⁺Foxp3⁺) was significantly reduced but was not restored by yolkin. The level of macrophages (CD14⁺CD169⁺) was not affected. In the mesenteric lymph nodes, a strong decrease in the CD19⁺ B cell number after CP treatment was found, but was not affected by yolkin treatment. Importantly, CD19⁺ B cells in the bone marrow were deeply depleted by CP, but were replenished by yolkin to the control level registered in untreated control mice.