*3.4. Assessment of Protein Expression by ELISA Confirms a Differential Expression of KRT19 and HSPD1 in the Lesional Skin of Female Psoriasis Patients*

Using ELISA, we analyzed the expression of KRT19 and HSPD1 in lesional skin as well as the PBMC of qPCR/ELISA psoriasis patients and healthy volunteers (Table 1). The performed analysis of skin samples revealed that the expression levels of both proteins were significantly higher in the lesional skin of patients compared to healthy volunteers (Figure 4a). Moreover, the expression levels of both proteins were significantly different (*p* < 0.05) in patients without menopause compared to patients with menopause. In contrast, we did not see significant differences in the expression of KRT19 and HSPD1 when we compared skin samples of non-menopausal and menopausal volunteers.

' ' **Figure 4.** The levels of the expression of estrogen-responsive proteins in clinical samples of psoriasis patients and healthy volunteers, assessed by ELISA. (**A**). The levels of gene expression in the samples of patients' lesional skin and in the skin of healthy volunteers. (**B**). The levels of gene expression in the PBMC obtained from the patients' blood and from the blood of healthy volunteers. The following individuals participated in these experiments: psoriasis patients without menopause (n = 10); psoriasis patients with menopause (n = 10); healthy volunteers without menopause (n = 6); and healthy volunteers with menopause (n = 5). \* *p* < 0.05 when women without menopause compared to women with menopause. # *p* < 0.05 when patients compared to healthy volunteers. Protein concentrations were measured in ng/mL.

' A similar analysis of the PBMC also revealed a significantly higher expression of HSPD1 in the lesional skin of patients compared to that of the healthy volunteers (Figure 4b). In contrast, changes in the expression of KRT19 were statistically insignificant although the expression level of KRT19 in patients' PBMC was elevated compared to the PBMC of healthy volunteers. In a similar manner, the expression level of HSPD1 in the PBMC of the patients without menopause was significantly higher compared to the patients with menopause, although we did not see significant differences when we analyzed changes in the expression of KRT19. In addition, there were no significant differences in the expression of both proteins between non-menopausal and menopausal healthy volunteers.

#### **4. Discussion**

– In this study, we analyzed skin samples of psoriasis patients using the LC–MS/MS method and reported of six ERPs, namely HMOX1, KRT19, LDHA, HSPD1, MAPK1, and CA2, that were differentially expressed in the lesional and uninvolved skin of female MS participants (Table 4) and were not present in the skin of their male counterparts (Table 5), suggesting an existence of a gender-dependent response to the disease. Using independent methods of analysis, namely qPCR and ELISA (Figures 3 and 4), we examined their expression in menopausal and non-menopausal female patients and the respective groups of healthy volunteers (Figure 3a,d). We also assessed the levels of sex hormones, namely ES, PG, and TS, in their blood (Figure 1).

In performing qPCR and ELISA experiments on a larger cohort of women, we confirmed that the identified ERGs and their encoding proteins were differentially expressed in female lesional skin compared to healthy skin (Figures 3a and 4a). In comparing nonmenopausal and menopausal patients, we found significant changes in the expression of *HMOX1*, *HSPD1*, *CA2*, and *MAPK1*. In contrast, the changes in their expression were insignificant when we compared non-menopausal and menopausal healthy volunteers. The results of the ELISA experiments were similar to the results of the qPCR analysis (Figure 4). Thus, the obtained data suggested that the observed changes in gene expression were caused by the differences in the levels of sex hormones and were associated with the disease because healthy volunteers did not have them. As we believe, the sex hormones, primarily E2, acted as modulators, altering the expression of ERGs and interfering with the disease.

This hypothesis is in agreement with our next finding. In performing the PCA of qPCR data, we found that a single factor was responsible for 53% of the variability between the samples (Figure 3a). Using K-mean clustering, we separated the samples in two groups that contained samples of qPCR/ELISA patients and samples of qPCR/ELISA volunteers. However, we could not achieve complete separation of individuals with and without menopause within those groups. Based on this finding, we proposed that in some women, changes in the expression of ERGs might not coincide with menopause. As we believe, deviations of this kind could be caused by a crosstalk of ES-activated signaling mechanisms and other signaling pathways (e.g., the pathways activated by proinflammatory cytokines).

In assessing the levels of sex hormones in the blood of qPCR/ELISA participants, we showed (Figure 1) that the blood of patients contained significantly less E2 and PG, and more TS compared to that of the healthy control. Moreover, menopausal patients and healthy volunteers contained less E2 and PG, and more TS compared to their nonmenopausal counterparts (Table 3). These results suggested that sex hormones influenced the gene expression in psoriatic skin. They also confirmed the previous findings [4,6,8] regarding that the fluctuations in the levels of E2, PG, and TS could potentially modulate the course of the disease.

In comparing gene expression profiles in lesional skin (Figure 3a) and the patterns of sex hormones in patients' blood (Table 3), we discovered significant changes between menopausal and non-menopausal patients, as well as between healthy volunteers and any group of patients. The expression levels of *HMOX1*, *KRT19*, *LDHA*, *HSPD1*, and *MAPK1* were higher in patients with menopause compared to patients without menopause. Moreover, same individuals had higher blood levels of E2 and PG (Table 3). In addition, the expression levels of *CA2* and blood levels of TS were higher in patients with menopause compared to patients without menopause.

Furthermore, when we combined two groups of healthy volunteers with and without menopause (Figure 3a) and considered them as one group, we saw differences between the expression profiles of ERGs in lesional skin (Figure 3a) and in the patterns of sex hormones in their blood (Table 3). The expression levels of *HMOX1*, *KRT19*, *LDHA*, *HSPD1*, and *MAPK1* were lower in healthy volunteers compared to any group of patients. In contrast, their blood levels of E2 and PG were higher compared to the same groups. In contrast, the expression levels of *CA2* and blood levels of TS in both groups of patients were lower compared to that of healthy volunteers.

Based on these findings, we concluded that sex hormones differentially contributed to the expression of individual ERGs. Presumably, E2 and PG made a greater contribution to the regulation of *HMOX1*, *KRT19*, *LDHA*, *HSPD1*, and *MAPK1*, whereas TS made a greater contribution to the regulation of *CA2*. However, additional experimental studies on cultured cells are needed to clarify the role of particular sex hormones in the regulation of individual ERGs primarily in the skin because the biological effects of sex hormones are tissue-specific and their regulation of gene expression is often presented as a cross-talk of several signaling pathways.

In comparing the expression of the identified ERGs in the skin (Figure 3a) and of the PBMC (Figure 3d), we found similarities in their expression profiles. In particular, the expression levels of *HMOX1*, *KRT19*, *LDHA*, and *HSPD1* were significantly higher in the PBMC of patients compared to healthy volunteers, in non-menopausal patients compared to non-menopausal volunteers, and in menopausal patients compared to menopausal volunteers. However, most of these changes were statistically insignificant. Based on these findings, we concluded that sex hormones, primarily E2, influenced gene expression in both types of samples in a similar way. This result was anticipated since psoriasis is a systemic disorder that targets various tissues and is associated with multiple comorbidities. Expectedly, the same mechanism was activated in response to the disease in both the skin and PBMC.

The previous studies showed that female sex hormones have significant immunomodulatory effects. In particular, ES reduces the production of macrophage-attracting cytokines (CXCL8, CXCL10, CCL2, -5, and -8) and the production of IL12 in keratinocytes. These cytokines, namely CXCL10, CCL5, and -8, recruit activated T cells [17], macrophages [18], and neutrophils [19], respectively. E2 inhibits the production of IL12 and TNF by dendritic cells. Moreover, it decreases the blood level of neutrophils. Based on these findings, we may consider low E2 level in the blood of our patients (Figure 1) as a potential risk for exacerbation of the disease.

PG stimulates the production of the Th<sup>2</sup> cytokines, namely IL4 and IL5, by T cells without altering the production of Th<sup>1</sup> cytokines [20]. Moreover, PG blocks androgen receptors (AR) and, in inhibiting the release of LH, reduces the level of circulating androgens (AG) in the blood. In fibroblasts, PG suppresses the transcription of *CXCL8* [21]. In contrast, when the PG level is low, TS activates ARs, contributing to the development of an inflammatory response [22]. These data suggest that the reduction in the PG level in the patients' blood that we observed in our study (Figure 1) could also contribute to the pathogenesis of the disease.

To our knowledge, this is the first study focused on gender-specific differences in gene and protein expression in psoriatic skin. Three of the six identified genes, namely *KRT19*, *LDHA*, and *HSPD1* were not previously associated with psoriasis. Depending on the role of the particular ERG in the pathogenesis of psoriasis, its differential expression may either promote or limit the growth of psoriatic plaques in a disease-affected area. Hemoxygenase 1 (HMOX1) is a stress protein. The expression of *HMOX1* can be induced by a variety of stimuli, including the proinflammatory cytokines TNF and IL17, which are abundant in the lesional skin of psoriasis patients [23]. The anti-inflammatory and anti-oxidant activities of HMOX1 are well-documented and can be considered as a part of a protective mechanism that contains (controls) the inflammatory response in lesional skin. The others already showed that inducers of *HMOX1* attenuate the inflammatory response in lesional skin [24]. Moreover, a proteolytic cleavage of HMOX1 generates several biologically active metabolites. One of them, the N-terminal peptide, binds to the promoter of *IL23A*, interfering with biological activities of proinflammatory cytokines IL12 and IL23 [25]. The others, bilirubin and CO, produce potent antioxidant and anti-inflammatory effects [26,27].

MAPK1 is one of two known extracellular signal-regulated kinases (ERKs). Although the role of MAPK1 in the inflammatory response, primarily for the induction of TNF [28], is well-documented, in a general sense, it is a signaling molecule that can be activated by multiple stimuli, including ES [29]. Due to the high sequence homology of ERKs and its similar role in the cell, two proteins, namely MAPK1/ERK2 and MAPK3/ERK1, are considered as two isoforms of the same enzyme. However, their expression patterns in lesional skin are different. MAPK3/ERK1 is strongly increased, whereas the expression

of MAPK1/ERK2 is slightly affected by the disease [30]. The cited paper also suggests that, similarly to HMOX1, ES is likely to contribute to the regulation of MAPK1 in lesional skin. Particularly, the involvement of ES would explain the strong induction of *MAPK1* in our female patients and answer the question concerning why the expression of *MAPK1* significantly decreases after menopause in patients (Figure 3a).

We also discovered that *LDHA*, which encodes LDH-M, the subunit of lactate dehydrogenase (LDH), was upregulated in lesional skin (Figure 3a). Compared to the other LDH subunits, LDH-M has a higher affinity to pyruvate. In other words, if lactate and pyruvate are equally available, LDH-M preferentially binds to pyruvate and converts pyruvate to lactate. It also oxidizes NADH, which is a coenzyme in this reaction, to NAD<sup>+</sup> [31]. According to the previously published data, *LDHA* is involved in several immunomodulatory processes that could potentially influence the flow of psoriasis. First, it decreases the proliferation of cytotoxic and other effector T cells and their production of cytokines because these cells become inactive at low glucose and high lactate concentrations in the medium [32]. Second, lactate activates a mechanism that stops the migration of T-cells, entrapping them in the inflamed area [33]. Third, a suppression of *LDHA* in macrophages reduces the phosphorylation of mitogen-activated protein kinase MAPK14/p38 [34]. At the same time, the accumulation of lactate in the inflamed tissue increases the production of IL17 by T cells [35]. In addition, *LDHA* is overexpressed in cancer cells that rely on aerobic glycolysis to maintain their higher proliferation and faster metabolic rates. For this reason, a higher expression of *LDHA* in lesional epidermis is also necessary due to hyperproliferation and acceleration of the metabolism in epidermal keratinocytes of psoriasis patients.

*HSPD1* encodes the mitochondrial chaperonin HSP60 that provides a favorable environment for the correct folding of unfolded and misfolded proteins. Similarly to *HMOX1*, *MAPK1*, and *LDHA*, *HSPD1* is induced in stress conditions. In cancer cells, a higher expression of *HSPD1* is needed due to their faster metabolic and proliferation rates, and more intensive protein trafficking between the cytoplasm and mitochondria [36]. Moreover, a higher expression of *HSPD1* in lesional skin, as we observed (Figure 3a), would help to maintain a faster turnover of the epidermal keratinocytes.

*KRT19* improves the rigidity of intermediate filaments [37]. Moreover, KRT19 activates NOTCH signaling pathways promoting nuclear translocation of β-catenin [38] and AKT [39]. The expression of *KRT19* in cancer cells increases their cell proliferation rate due to its ability to stabilize cyclin D3 [38]. A similar effect is observed in HaCaT cells that stop expressing *KRT19* before reaching the confluence [40]. We presume the upregulation of KRT19 in lesional skin helps epidermal keratinocytes to adapt to higher proliferation and metabolic rates in the areas affected by the disease.

*CA2*, which we also identified in our study, exhibits a higher catalytic rate for the conversion of bicarbonate to carbon dioxide compared to the other carbonic anhydrases. This prevents acidification of the cytoplasm. In the opposite direction, CA2 facilitates the diffusion of carbon dioxide through the cytoplasm, converting carbon dioxide to bicarbonate. According to our observations (Figure 3a), *CA2* levels were decreased in patients with menopause compared to either of the three groups, namely patients without menopause, healthy volunteers without menopause and healthy volunteers with menopause, suggesting that the downregulation of *CA2* in menopausal qPCR/ELISA patients (Figure 3a) could be caused by an action of an unidentified factor, such as TS which is capable of interfering with ES [41] and can be suppressed by PG [22].

The differential expression of the identified ERGs can be part of an adaptive mechanism (Figure 5) that protects the skin cells from chronic inflammatory responses. Presumably, this mechanism is more efficient in female patients without menopause since they have higher levels of female sex hormones. Cooperating with several transcription factors, ES modulates the responses to various stimuli such as oxidative stress [42] and hypoxia [43]. For instance, there is an abundance of evidence regarding synergism between ES and hypoxia-induced factor 1α (HIF1α), which is highly unstable under normoxic conditions [44]. However, HIF1α becomes more stable in lesional skin [45] because lactate, the

preferential product of LDH-M, stabilizes HIF1α by inhibiting prolyl hydroxylase (PHD2). It also triggers the nuclear translocation of HIF1α and the following induction of hypoxia responsive genes [46]. In contrast, the inhibition of LDHA causes a rapid degradation of HIF1α in proteasomes [47]. Many genes, including ones that we mentioned above, are common targets of ES and HIF1α [44,48]. Respectively, their expression (Figure 5) also depends on both ES and HIF1α [49]. It can be suppressed by either ERα antagonists [44] or inhibitors of ERα or HIF1α, as well as by HIF1α destabilizing factors. HIF1α in proteasomes common targets of ES and HIF1α depends on both ES and HIF1α . It can be suppressed by either ERα antagonists or inhibitors of ERα or HIF1α HIF1α destabilizing fa

 <sup>−</sup> **Figure 5.** Regulation of estrogen-responsive proteins in lesional skin of female psoriasis patients with and without menopause: the proposed model. The signs ↑ and ↓ indicate that the protein expression is increased and decreased, respectively. The sign ⇆ indicates that chemical reaction between HCO<sup>3</sup> <sup>−</sup> and can be reversed.

The observed changes in the levels of sex hormones (Table 3) are of high clinical value. According to us (Figure 1) and others [22], low levels of sex hormones in the blood of women with psoriasis have a significant impact on the disease. Pregnant patients with moderate or severe forms of psoriasis have a higher risk of abortion, eclampsia, premature rupture of membranes, and macrosomia [50]. Another serious problem associated with psoriasis is female infertility [51]. However, it is still hard to say whether these complications are caused by psoriasis or comorbidities associated with the disease. For this reason, we explored the molecular basis of ES action in psoriatic skin and identified that there are six differentially expressed ERGs.

A medical correction of sex hormones may have a significant impact on the disease. The previously published data suggest that ES containing oral contraceptives [8] and hormone replacement therapy [52] may improve psoriasis. Conversely, it is well-documented that synthetic ES, if it is used as a therapeutic agent, can cause serious side effects such as cardiovascular events, thromboembolic disease, and breast cancer [53,54]. In this regard, knowledge of ERGs controlled by sex hormones in lesional skin suggests new hormone-free options to stabilize their expression in lesional skin, such as inducers of gene expression, biologically active peptides, and allosteric activators of their enzyme activity. Presumably, these potential treatment options may also help to avoid serious adverse effects.

In conclusion, a deficiency of female sex hormones seems to be among the risk factors that influence the flow of psoriasis in women. Respectively, the maintenance of their

–

normal (physiological) levels may potentially prevent or suppress the disease. A detailed analysis of the proteins identified in the LC–MS/MS study, namely HMOX1, KRT19, LDHA, HSPD1, MAPK1, and CA2, revealed that they were differentially expressed in female lesional skin and were not present in male lesional skin. Using qPCR and ELISA, we found that the levels of their expression were higher in younger participants that were not in menopause. According to us and others, these proteins can be part of an adaptive mechanism that protects skin cells from the developing inflammatory response. In providing new insight on the molecular basis of ES signaling in female patients, we propose that ES mediates its anti-inflammatory effects in lesional skin, synergistically interacting with hormones/transcription factors that contribute to the stress response.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jpm11090925/s1, Table S1: Excel-file with heat map that describes changes in protein expression in the skin of the male and female individuals that participated in the LC–MS/MS study and the results of the statistical analysis in male (n = 3) and female psoriasis patients (n = 2) compared to healthy volunteers (n = 5). The differences in protein expression were analyzed using the Mann– Whitney U test.

**Author Contributions:** Conceptualization, V.S.; Data curation, A.M.; Investigation, V.S. and A.S.; Project administration, V.S. and I.K.; Resources, E.D. (Elena Denisova), M.D., E.D. (Eugenia Dvoryankova), O.V.Z., E.S. and N.P.; Supervision, V.S.; Validation, A.M.; Visualization, A.M.; Writing—original draft, A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (Research Ethics Committees of Western Norway HR 2020-0870). The participants were informed of the nature of the study, gave their informed consent, and were told they could voluntary withdraw during the interviews at any time.

**Informed Consent Statement:** Written informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

**Data Availability Statement:** The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository [15] with the dataset identifier PXD021673.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

