*3.3. IgY Treatment-Induced Changes in Lymphocyte Distribution in Peripheral Blood and Spleen Cell Suspensions in Experimental Murine Model of Psoriatic Dermatitis*

To evaluate the immune cells populations/subpopulations that follow the clinical improvement of the induced psoriasis lymphocyte immunophenotyping was performed by flow cytometry from both peripheral blood and spleen cell suspensions. For all experimental groups we quantified T lymphocytes (CD3ε + ), with T-helper (CD4+CD8−) and T-suppressor/cytotoxic (CD8a+CD4−) subsets, B lymphocytes (CD3ε <sup>−</sup>CD19<sup>+</sup> ) and NK cells (CD3ε <sup>−</sup>NK1.1<sup>+</sup> ).

ε − <sup>−</sup> ε − Therefore, a statistically significant lower percentages of T-CD4+ (*p* = 0.007) and signifi-cantly increased of T-CD8a+ lymphocytes (*p* = 0.007 vs) were obtained (Figure 4a). As a consequence of the changes observed in T subsets distribution, the T-CD4+/T-CD8+ ratio was decreased in Ps group as compared to control group (*p* = 0.003) (Figure 5a). Also, a decreased percentage of B lymphocytes (*p* = 1.1 × 10−<sup>6</sup> ) and a significantly increased of NK1.1+ cells percentages (*p* = 0.0001) were registered (Figure 6a).

ε − The main changes observed in spleen cell suspensions were statistically significant in Ps group, namely lower percentages of T-CD4<sup>+</sup> (*p* = 0.02) and B lymphocytes (*p* = 4 × 10−<sup>7</sup> ), (Figures 4b and 6b). T-CD4+/T-CD8<sup>+</sup> ratio is decreased in Ps mice as compared to control group but not statistically significant (Figure 5b).

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ε **Figure 4.** Distribution of T-CD4<sup>+</sup> and T-CD8a<sup>+</sup> lymphocytes in peripheral blood and spleen cell suspension. (**a**) Peripheral blood-distribution of T-CD4<sup>+</sup> and T-CD8a<sup>+</sup> lymphocytes for IgY-treated Ps group (*n* = 12) (53 ± 3.6 and 42 ± 3.3) as compared to naturally remitted Ps group (*n* = 8) (53 ± 3 and 39 ± 2.4), control group (*n* = 8) (53 ± 4.7 and 41 ± 1.8) and Ps group (*n* = 8) (47 ± 1.5 and 46 ± 2.8); (**b**) Spleen cell suspension-distribution of T-CD4<sup>+</sup> and T-CD8a<sup>+</sup> lymphocytes for IgY-treated Ps group (*n* = 12) (48 ± 2.2 and 41 ± 2.3) as compared to naturally remitted Ps group (*n* = 8) (52 ± 1.5 and 39 ± 1.8), control group (*n* = 8) (56 ± 4.3 and 38 ± 6.1) and Ps group (*n* = 8) (46 ± 8.1 and 40 ± 1.9). The results (% of CD3ε + lymphocytes) are presented as mean ± SD; *n* = number of mice).

**Figure 5.** Distribution of T-CD4+/T-CD8a<sup>+</sup> ratio in peripheral blood and spleen cell suspension. (**a**) Peripheral blooddistribution of T-CD4+/T-CD8a<sup>+</sup> ratio for IgY-treated Ps group (*n* = 12) (1.25 ± 0.2) as compared to naturally remitted Ps group (*n* = 8) (1.37 ± 0.1), control group (*n* = 8) (1.29 ± 0.2) and Ps group (*n* = 8) (1.04 ± 0.1); (**b**) Spleen cell suspension– distribution of T-CD4+/T-CD8a<sup>+</sup> ratio for IgY-treated Ps group (*n* = 12) (1.17 ± 0.1) as compared to naturally remitted Ps group (*n* = 8) (1.35 ± 0.1), control group (*n* = 8) (1.52 ± 0.4) and Ps group (*n* = 8) (1.15 ± 0.2). The results are presented as mean ± SD; *n* = number of mice).

**Figure 6.** Distribution of B-CD19<sup>+</sup> and NK1.1<sup>+</sup> cells in peripheral blood and spleen cell suspension. (**a**) Peripheral blooddistribution of B-CD19<sup>+</sup> and NK1.1<sup>+</sup> cells for IgY-treated Ps group (*n* = 12) (83 ± 3.2 and 4 ± 0.7) as compared to naturally remitted Ps group (*n* = 8) (77 ± 5.5 and 5 ± 1.1), control group (*n* = 8) (85 ± 5.3 and 4 ± 0.7) and Ps group (*n* = 8) (31 ± 12.8 and 12 ± 3.3); (**b**) Spleen cell suspension–distribution of B-CD19<sup>+</sup> and NK1.1<sup>+</sup> cells for IgY-treated Ps group (80 ± 1 and 3 ± 0.4) as compared to naturally remitted Ps group (70 ± 2.1 and 5 ± 0.5), control group (83 ± 3.6 and 6 ± 0.8) and Ps group (40 ± 9 and 5 ± 1.8). The results (% of CD3ε <sup>−</sup> lymphocytes) are presented as mean ± SD; *n* = number of mice).

Analysis of T-CD4<sup>+</sup> and T-CD8a<sup>+</sup> lymphocyte subsets in peripheral blood revealed normalization of mean percentage values of these parameters for both IgY-treated Ps group and naturally remitted Ps group (Figure 4a). There were statistically significant differences between the values of T-CD4<sup>+</sup> and T-CD8a<sup>+</sup> subsets for Ps group and IgY-treated Ps group (*p* = 0.002 for T-CD4<sup>+</sup> and *p* = 0.04 for T-CD8a<sup>+</sup> ), and naturally remitted Ps group (*p* = 0.002 for T-CD4<sup>+</sup> and *p* = 0.006 for T-CD8a<sup>+</sup> ), respectively, comparable to the differences observed between Ps group and controls for the investigated parameters. No statistically significant differences were observed between IgY-treated Ps group and naturally remitted Ps group for these T-subsets. Furthermore, no statistically significant differences were observed between IgY-treated Ps group and control group for T-CD4<sup>+</sup> and T-CD8a<sup>+</sup> subsets (*p* > 0.05), thus statistically underlining the normalization of these values after IgY treatment.

Data obtained for T-CD4<sup>+</sup> and T-CD8a<sup>+</sup> subpopulations in spleen cell suspensions revealed a tendency to normalization for both IgY-treated Ps group and naturally remitted Ps group (Figure 4b). For T-CD8a<sup>+</sup> lymphocytes, no statistically significant differences were observed between the IgY-treated Ps group and control group. Although the values of T-CD4<sup>+</sup> subset obtained for IgY-treated Ps group were significantly lower than control (*p* = 0.006), no statistically significant differences were observed between the IgY-treated Ps group and naturally remitted Ps group for T-CD4<sup>+</sup> and T-CD8a<sup>+</sup> subsets.

Analysis of the T-CD4+/T-CD8a<sup>+</sup> ratio in the peripheral blood revealed the normalization of the mean values for both IgY-treated Ps group and naturally remitted Ps group (Figure 5a). There were statistically significant differences between Ps group and naturally remitted Ps groups (*p* = 0.0003), respectively IgY-treated Ps group (*p* = 0.01), and no statistically significant differences between control group and IgY-treated Ps group, respectively naturally remitted Ps group. The value of the T-CD4+/T-CD8a<sup>+</sup> ratio for IgY-treated group was almost identical to control group (1.25 vs. 1.29).

A tendency of normalization of T-CD4+/T-CD8a<sup>+</sup> ratio values was also noticed in spleen cell suspensions for both experimental groups. Although the values obtained for IgYtreated Ps group were significantly lower than control (*p* = 0.04), no statistically significant

differences were observed between the IgY-treated Ps group and naturally remitted Ps group for T-CD4+/T-CD8a<sup>+</sup> ratio.

Analysis of B-CD19<sup>+</sup> and NK1.1<sup>+</sup> cells in peripheral blood revealed normalization of these parameters for both IgY-treated Ps group and naturally remitted Ps group (Figure 6a). Statistically significant differences were observed between the Ps group and the naturally remitted Ps group (*p* = 0.0003 for B-CD19<sup>+</sup> and *p* = 0.01 for NK1.1<sup>+</sup> ), respectively, and the IgY-treated Ps group (*p* = 1 × 10−<sup>8</sup> for B-CD19<sup>+</sup> and *p* = 9.8 × 10−<sup>6</sup> for NK1.1<sup>+</sup> ). Although for both experimental groups B-CD19<sup>+</sup> and NK1.1<sup>+</sup> normalization in peripheral blood was noticed, it is important that the values normalization obtained for IgY-treated Ps group is more pronounced, and the days necessary for skin healing is reduced compared to the naturally remitted Ps group. The normalization of values for the IgY-treated group is also supported by the fact that no statistically significant differences were obtained between the IgY-treated group and controls.

The main change observed in the cellular population of the spleen was the normalizing of B-CD19<sup>+</sup> cells revealed by a significant increase of B lymphocyte percentages in both IgY-treated Ps group and naturally remitted Ps group (Figure 6b). Statistically significant differences were observed between Ps group and the naturally remitted Ps group (*p* = 0.0006), respectively IgY-treated Ps (*p* = 2.1 × 10−<sup>9</sup> ) group, differences were also noticed between the Ps group and controls (*p* = 4 × 10−<sup>7</sup> ). Although for both experimental groups, the normalization of B-CD19<sup>+</sup> values from spleen cell suspension were observed, once more the normalization is enhanced for IgY-treated Ps group, and less healing days necessary compared to naturally remitted Ps group. No statistically significant differences were obtained for B-CD19<sup>+</sup> lymphocytes between IgY-treated Ps group and controls (*p* > 0.05), the values obtained being comparable. For NK1.1<sup>+</sup> cells, there is a tendency to normalize their values, and it was more pronounced in this case for the naturally remitted Ps group, but no statistically significant differences were observed between the IgY-treated Ps group and naturally remitted Ps group.
