*3.4. IgY Treatment-Induced Changes in NK Phenotype in Peripheral Blood and Spleen Cell Suspensions in Experimental Murine Model of Psoriatic Dermatitis*

The expressions on NK cells of CD49b, CD11b, CD43, CD27, KLRG1-maturation markers, on CD69, CD28, CD11c, NKp46-activation markers, respectively, were quantified for all experimental groups.

The analysis of maturation markers in peripheral blood (Figure 7) showed a significant tendency to increase their expression on NK cells as compared to control group and the differences between the experimental groups were statistically significant (*p* = 0.01; *p* = 0.0009; *p* = 0.01). The level of CD49b on NK cells is significant reduced in Ps group. The percentages of NK1.1+CD11b<sup>+</sup> cells in Ps group are higher than controls, but without statistical significance. In spleen cell suspension (Figure 8), analysis of maturation markers revealed the same tendency of variation: increased values for CD11b, CD27, KLRG1 levels on NK cells and lower values for CD49b and CD43 in Ps mice as compared to controls. Only for CD49b and KLRG1 the differences were statistically significant (*p* = 0.01; *p* = 0.008).

Analysis of CD69, CD11c and CD28 (activation markers) on NK1.1<sup>+</sup> cells in peripheral blood revealed significantly increased values in Ps group *p* = 8.5 × 10−11; *p* = 9.5 × 10−<sup>9</sup> ; *p* = 0.003) compared to controls; the expression of NKp46 on NK1.1<sup>+</sup> cells is lower in Ps mice as compared to controls and the differences between the experimental groups were statistically significant (*p* = 0.001) (Figure 9). We found the same tendency of variation for activation markers in spleen cell suspensions (Figure 10): significantly increased values for CD69, CD11c and CD28 in Ps group (*p* = 4.1 × 10−12; *p* = 2.1 × 10−<sup>7</sup> ; *p* = 0.0001) compared to controls; the expression of NKp46 on NK1.1<sup>+</sup> cells is lower in Ps mice as compared to controls, and the differences between the experimental groups were statistically significant (*p* = 0.0003).

**Figure 7.** Expression of maturation markers on NK cells in peripheral blood. Expression of CD49b, CD11b, CD43, CD27 and KLRG1 levels on NK1.1<sup>+</sup> cells for IgY-treated Ps group (*n* = 12) (72 ± 4.7; 85 ± 2.7; 89 ± 4.6; 16 ± 6.1; 49 ± 3.7) as compared to naturally remitted Ps group (*n* = 8) (61 ± 8.3; 93 ± 1.8; 92 ± 2.1; 27 ± 0.8; 67 ± 4.1), control group (*n* = 8) (71 ± 11; 93 ± 1.5; 94 ± 2.7; 15 ± 3; 63 ± 6) and Ps group (*n* = 8) (56 ± 6.5; 94 ± 1.8; 97 ± 1.3; 24 ± 4.1; 73 ± 6.6). The results (% of NK1.1<sup>+</sup> cells) are presented as mean ± SD; *n* = number of mice).

**Figure 8.** Expression of maturation markers on NK cells in spleen cell suspensions. Expression of CD49b, CD11b, CD43, CD27 and KLRG1 levels on NK1.1<sup>+</sup> cells for IgY-treated Ps group (*n* = 12) (61 ± 3.7; 75 ± 3.1; 73 ± 6.8; 28 ± 3.9; 39 ± 3.9) as compared to naturally remitted Ps group (*n* = 8) (66 ± 0.2; 72 ± 3.2; 82 ± 4.1; 31 ± 3.1; 47 ± 3.7), control group (*n* = 8) (70 ± 5.1; 73 ± 1.4; 82 ± 2.3; 30 ± 2.9; 41 ± 4.3) and Ps group (*n* = 8) (48 ± 13.9; 77 ± 5.2; 78 ± 6; 36 ± 7.8; 48 ± 5.4). The results (% of NK1.1<sup>+</sup> cells) are presented as mean ± SD; *n* = number of mice).

Analysis of NK cell maturation markers in peripheral blood (Figure 7) revealed a normalization of values for NK1.1+CD49<sup>+</sup> and NK1.1+CD27<sup>+</sup> cells in the IgY-treated Ps group. As there are statistically significant differences (*p* = 0.01 and *p* = 0.0009) obtained for these parameters between the Ps and control groups, after application of the IgY treatment we did not find statistical differences when comparing the values obtained for IgY-treated Ps group and controls. The expression of CD11b, CD43 and KLRG1 markers on NK1.1<sup>+</sup> cells is significantly lower for IgY-treated Ps group as compared to control group. For the naturally remitted Ps group, the values for CD49b, CD11b, CD43 and KLRG1 are normalized when comparing the values obtained for naturally remitted Ps group and controls. The expression of CD27 on NK1.1<sup>+</sup> cells was still significantly increased (*p* = 0.0003) for the naturally remitted Ps group, being almost equal to that obtained for the Ps group.

**Figure 9.** Expression of activation markers on NK cells in peripheral blood. Expression of CD69, CD11c, CD28 and NKp46 levels on NK1.1<sup>+</sup> cells for IgY-treated Ps group (*n* = 12) (31 ± 4; 36 ± 3.8; 1 ± 0.5; 92 ± 4.2) as compared to naturally remitted Ps group (*n* = 8) (14 ± 1.6; 43 ± 6.4; 2 ± 0.3; 97 ± 0.6), control group (*n* = 8) (13 ± 2.6; 27 ± 3.7; 1.2 ± 1.2; 97 ± 0.5) and Ps group (*n* = 8) (73 ± 5.7; 81 ± 8.1; 4 ± 1.5; 92 ± 3.2). The results (% of NK1.1<sup>+</sup> cells) are presented as mean ± SD; *n* = number of mice).

**Figure 10.** Expression of activation markers on NK cells in spleen cell suspensions. Expression of CD69, CD11c, CD28 and NKp46 levels on NK1.1<sup>+</sup> cells for IgY-treated Ps group (*n* = 12) (13 ± 3.8; 33 ± 6.2; 3 ± 1.2; 79 ± 5) as compared to naturally remitted Ps group (*n* = 8) (12 ± 2.2; 39 ± 8.1; 3 ± 1.8; 83 ± 3.2), control group (*n* = 8) (4 ± 3.5; 26 ± 7.1; 1 ± 0.7; 75 ± 5.7) and Ps group (*n* = 8) (65 ± 3.5; 74 ± 8.2; 29 ± 11.5; 61 ± 6.8). The results (% of NK1.1<sup>+</sup> cells) are presented as mean ± SD; *n* = number of mice).

In the spleen (Figure 8), the analysis of CD11b, CD27 and KLRG1 maturation markers revealed the normalization of their expression on NK cells following IgY treatment when comparing to control group. CD43 expression on NK cells decreased after IgY treatment. There were no statistically significant differences between IgY-treated Ps group and naturally remitted Ps group for CD49b, CD11b, CD43 and CD27 on NK cells.

Analysis of the expression of activation markers on NK1.1<sup>+</sup> cells in peripheral blood after IgY treatment revealed the normalization of CD28 values when compared to controls. For CD69 and CD11c levels in IgY-treated Ps group we observed a significant decreasing trend compared to Ps group but the expressions of these NK markers are significantly increased compared to control group (Figure 9). Although there is no statistically significant difference between the IgY-treated Ps group and naturally remitted Ps group for NKP46 expression, its expression on NK cells after IgY treatment is comparable to Ps group,

namely below normal limits. For naturally remitted Ps group, the values of all activation markers have normalized when comparing to controls, except for CD11c, whose expression is significantly increased compared to control group and IgY-treated Ps group (*p* = 0.001 and *p* = 0.03, respectively).

Analysis of the expression of CD69, CD11c and CD28 activation markers on NK1.1<sup>+</sup> cells in spleen showed a pronounced decreasing trend for both IgY-treated mice and naturally remitted Ps group, toward normalization of their values (Figure 10). For CD11c expression, there is no statistically significant difference between IgY-treated Ps group and control group, while for naturally remitted Ps group, there are still significant differences (*p* = 0.03), when compared to controls. For all activation markers there are no statistically significant difference between IgY-treated Ps group and naturally remitted Ps group. NKp46 expression on NK cells have normalized in both IgY-treated group and naturally remitted Ps group as compared to the control group. Normalization of NKp46 values is more evident after IgY treatment.
