**4. Discussion**

Psoriasis affecting the health of numerous individuals world-wide has a multifactorial pathogenesis and the exact triggering factor remains still unclear, As the skin is the major human organ with multiple functions, Ps instalment would trigger complex systemic disturbances. Alterations in the skin and intestinal microbiome are involved in the pathogenesis of psoriasis, therefore microbiome restoration becomes a promising preventive/therapy strategy in psoriasis [49].

Several years ago, it was shown that the overall microbial diversity is increased in the psoriatic plaque [50]. More recent studies proclaim an abnormal gut/skin microbiome as a potential driving force of systemic inflammation underlying Ps. It is hypothesized a gut-skin axis to be involved in Ps etiology as gut microbiota dysbiosis may alter systemic immunity and diminishes skin's physiological functions [42,51]. Regarding therapy strategies, Ps treatment resembles bowel disease and could implicate appropriate antibiotics to restore a normal flora, and also the use of prebiotics might be an alternative avenue to explore [52]. Therefore, aiding current therapies with adjuvant compounds becomes a necessity. As IgY is gaining new therapeutical potential in the anti-viral and anti-bacterial fight and acknowledging all the accumulated data, we have initiated in a psoriasis experimental model an adjuvant therapy using oral IgY developed against several pathogenic bacteria to evaluate the potency to alleviate the psoriatic lesions and to restore the cellular immune-related mechanisms.

The IMQ-induced psoriasiform dermatitis model [47] represents one of the most used inducible systems in studying Ps due to its reduced cost, rapid induction of skin inflammation and high reproducibility. Topical application of IMQ in animal models induce the formation of cutaneous lesions similar with human Ps plaque, [53–55]. group [53,54], Splenomegaly, as an indicator of intense lymphocyte activation, was observed in all experimental groups in which psoriatic dermatitis was induced. As recently published, splenomegaly is a characteristic of this animal model [56] and it is an indicator that although the induction of lesions was topical there is a systemic immune response. Following oral therapy with IgY or naturally healing, the values of spleen weight and SW / BW ratio were identical to the control group values. Practically, the splenomegaly installed after 6 consecutive days of IMQ-based cream topical application was completely remitted, for both experimental groups.

As reported in Ps patients [57], peripheral and spleen immune cell deregulations were found. As previously reported also by other groups [58] lower percentages of T-CD4<sup>+</sup> and B lymphocytes, while the percentages of T-CD8a<sup>+</sup> lymphocytes and NK1.1<sup>+</sup> cells were significantly increased. As a consequence, the T-CD4+/T-CD8<sup>+</sup> ratio was significant decreased in Ps mice. The main changes observed in spleen cell suspensions were statistically significant, namely lower percentages of T-CD4<sup>+</sup> and B lymphocytes for Ps group as compared to controls. T-CD4+/T-CD8<sup>+</sup> ratio is decreased in Ps mice, but the differences

between the experimental groups were not statistically significant. The values obtained for these immunological parameters are comparable to the results published by our research team for psoriatic dermatitis mice model in which the IMQ-based cream was applied for 5 consecutive days [44] and with other group's results [59].

Analysis of T-CD4<sup>+</sup> and T-CD8a<sup>+</sup> lymphocyte subsets in peripheral blood revealed normalization of these parameters for IgY-treated Ps, naturally remitted Ps and control groups. T-CD8a<sup>+</sup> lymphocytes, identified in spleen cell suspensions in the IgY-treated Ps group are identical to the 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> subset. Recent findings have shown that T-CD8<sup>+</sup> cells are involved in psoriasiform skin inflammation and that memory T cells are involved in the pathogenesis of psoriasis, especially its recurrence. Therefore, normalization of these values brings clear clinical benefit [60].

As expected, the T-CD4+/T-CD8a<sup>+</sup> ratio in peripheral blood also revealed the normalization pattern of IgY-treated Ps group compared to naturally remitted Ps or control group. A tendency of normalization was also noticed in spleen cell suspensions for both experimental groups. Although T-CD4+/T-CD8a<sup>+</sup> ratio for IgY-treated 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.

Even though B-CD19<sup>+</sup> and NK1.1<sup>+</sup> normalization in peripheral blood was noticed the normalization for IgY-treated Ps group is more pronounced.

The main change observed in the spleen cell suspension was the normalizing of B–CD19<sup>+</sup> cells by significant increase of B lymphocyte percentages in both IgY-treated Ps group and naturally remitted Ps group. B cells have an important role in the protection against different infectious and inflammatory diseases, but there are very few reports on B lymphocytes involvement in Ps. The regulatory sub-population of B cells, Bregs were found decreased in Ps patients [61] and moreover, it was shown that Bregs may positively influence the course of Ps by producing IL-10 [62,63]. Therefore, the B cells increase that we have noticed in the treated group could account for the clinical improvement of the induced Ps.

For NK1.1<sup>+</sup> cells, there is a tendency to normalize the values, in the naturally remitted Ps group, with no statistically significant differences when compared to IgY-treated Ps group. The role of NK cells in Ps development is not fully elucidated. Although NK cells are recruited in human psoriatic lesions and in the induced Ps in mice, the studies regarding NK cells involvement in Ps do not abound [64]. The level of maturation marker CD49b on NK cells is significantly reduced in the Ps group. In spleen cell suspension, 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.

Analysis of activation markers CD69, CD11c and CD28 on NK1.1<sup>+</sup> cells in peripheral blood revealed significantly increased values in Ps group 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. Published studies report controversial results regarding NK cells in Ps and the matter is still subject of debate [65–67].

We found the same tendency of variation for activation markers in spleen cell suspensions, namely significantly increased values for CD69, CD11c and CD28 in Ps group compared to controls along with decreased expression of NKp46 on NK1.1+, with statistically significant differences. As a major activating receptor, NKp46, is an NK cell specific surface marker involved in all NK physiological immune processes [68] therefore an indicator that NK cells are mis-functioning due to the induced Ps.

In IgY-treated Ps group, NK cell maturation markers assessed in the peripheral blood revealed a normalization of values for NK1.1+CD49<sup>+</sup> and NK1.1+CD27<sup>+</sup> cells. Thus, the statistically significant differences obtained for these parameters between the Ps and control groups subside after IgY treatment. For naturally remitted Ps group, the values for CD49b, CD11b, CD43 and KLRG1 are as well normalized when compared to controls. As already mentioned, it should be noted that the healing period was longer for naturally remitted Ps group compared to the IgY-treated group. The expression of CD27 on NK1.1<sup>+</sup> cells was still significantly increased for the naturally remitted Ps group, being almost equal to that obtained for Ps group. NK maturation in periphery is characterized by an upregulation of CD11b, CD43, KLRG1, and Ly49 receptors, and a downregulation of CD27 [69,70], therefore we can speculate that even in clinically remitted psoriatic lesions the NK population remains alert to any psoriatic-dependent antigen.

In spleen cell suspension, analysis of CD11b, CD27 and KLRG1 maturation markers revealed the normalization of their expression on NK cells following IgY treatment compared to the control group. CD43 expression on NK cells decreased after IgY treatment. In contrast to the periphery, in the spleen, no statistically significant differences between IgY-treated Ps group and naturally remitted Ps group for CD49b, CD11b, CD43 and CD27 on NK cells was found. Yet again we can speculate that while NK residing in the secondary immune organs, such as the spleen, have normalized their parameters while in the periphery there are still populations that patrol in search of a psoriatic-like antigen.

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 markers on NK cells are significantly increased compared to control group. Although there is no statistically significant difference between IgY-treated Ps group and naturally remitted Ps group for NKP46, its expression on NK cells after IgY treatment is comparable to the Ps group, namely below normal limits. For naturally remitted Ps group, the values of all activation markers have normalized, except for CD11c, whose expression is significantly increased compared to control group and IgY-treated Ps group. Several years ago, it was reported that the CD56+CD16+CD11c<sup>+</sup> NK population are endowed with important characteristics such as IFN-γ production, tumor cell cytotoxicity and promotion of *γδ* T lymphocyte proliferation [71]. Therefore, in our experimental model NK cells retain their activation capacity as proven by CD11c expression.

In spleen cell suspensions analysis of the expression of CD69, CD11c and CD28 activation markers on NK1.1<sup>+</sup> cells showed a pronounced decreasing trend for IgY-treated mice normalizing their values. For CD11c expression, there is no statistically significant difference between IgY-treated Ps group and control group (*p* > 0.05), while for naturally remitted Ps group, there are still significant differences. 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 obvious after IgY treatment.

Study limitations. We acknowledge some limitations of our study. Thus, as human Ps is a complex auto-immune disease comprising, as presented, various triggering factors, the mice model is an induced one, therefore it misses probably more complex relations gut-skin interrelation. Another limitation of the study is that the IgY compound is designed for human ingestion as it is comprised out of IgY developed against antibiotic-resistant bacteria, therefore there could be bacterial strains that were missed in our mice model. Yet, in our model the Ps lesions subside earlier than the naturally remitted group probably as the utilized IgY is most probably restoring the digestive track microbiota. This limitation is overridden by the findings that mouse and human gut microbiota have similarity at the genus level [72]. Moreover, the mouse gut microbiota has similar functionality with the human one [73,74], therefore we can speculate in our experimental model that at least for

some of the gut bacteria the IgY compound restored the microbiota inducing hence the solving of the induced psoriasis.

Perspectives. Far from being exhaustive, our work can open new perspective in Ps therapy. Therefore, we can foresee application in human psoriasis by first establishing the patients gut microbiota, then inoculating hens with the bacteria and isolating the raised IgY. Then, in conjunction with the standard psoriasis therapy, purified IgY can be ingested in doses matching the ones tested within our work. As shown, IgY preparations are nonallergenic and have high biocompatibility. Hence, one can imagine in the future adjuvant setting in which personalized IgY could aid the established therapy, alleviate the psoriatic lesions and improve the overall health status of the patient.
