*3.2. LC–MS/MS Study Identifies Six Estrogen-Responsive Proteins That Are Differentially Expressed in Male and Female Psoriatic Skin*

To identify a possible gender-specific response to the disease, we analyzed skin samples donated by MS psoriasis patients (n = 5) and MS healthy volunteers (n = 5) of both genders using LC/MS–MS. The analysis demonstrated that 756 proteins were differentially expressed in patients' lesional and uninvolved skin. The distribution of DEPs between the groups of samples is shown on a Venn diagram (Figure 2a). Samples of male lesional and uninvolved skin (n = 3) contained 479 and 128 DEPs (Figure 2b) compared to the skin of healthy volunteers (n = 5). Samples of female lesional and uninvolved skin (n = 2) contained 419 and 111 DEPs (Figure 2b) compared to the skin of healthy volunteers. Their paired comparison revealed 123 proteins that were differentially expressed in female skin and were not present in male skin (Supplementary Table S1). Particularly, 26 and 86 proteins were differentially expressed in female uninvolved and lesional skin. Moreover, 11 proteins were differentially expressed in both groups of samples (Figure 2b).

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– – **Figure 2.** Venn diagram comparing DEPs in the donated skin samples as assessed by LC/MS–MS analysis. (**A**) Paired comparison of the samples obtained from lesional and uninvolved skin of the same psoriasis patients (n = 5) and skin of healthy volunteers (n = 5). (**B**) Analysis of gender-specific changes in protein expression. Samples of male (n = 3) and female (n = 2) psoriatic skin were compared to the skin of healthy volunteers (n = 5). The numbers indicated in the diagram are the numbers of DEPs in the compared groups of samples (*p* < 0.05). DEPs chosen for PO analysis are encircled. The data were compared using the Mann–Whitney U test.

The following protein ontology analysis of 123 proteins differentially expressed in female lesional and uninvolved skin performed on GO terms revealed 14 overrepresented biological processes (Table 4) including GO:0043627, which is the response to estrogen enriched by six DEPs, namely HMOX1, KRT19, LDHA, HSPD1, MAPK1, and CA2 (*p* = 0.005; FDR = 0.005). Four identified proteins, namely HMOX1, KRT19, LDHA, and MAPK1, were differentially expressed in female lesional skin, whereas HSPD1 and CA2 were also differentially expressed in female uninvolved skin. Among the mentioned six DEPs, CA2 was the only less abundant protein in patients' samples, whereas the others were more abundant in patients' skin samples compared to healthy skin. A similar analysis of the proteins differentially expressed in male lesional and uninvolved skin and were not differentially expressed in lesional and uninvolved female skin (Supplementary Table S1) revealed 11 overrepresented biological processes (Table 5).


**Table 4.** The ontology analysis of proteins differentially expressed in female lesional and uninvolved skin. The analyzed proteins were differentially expressed in female lesional and uninvolved skin and were not differentially expressed in male lesional and uninvolved skincompared to the skin of healthy volunteers.


**Table 4.** *Cont.*

**Table 5.** The ontology analysis of proteins differentially expressed in male lesional and uninvolved skin. The analyzed proteins were differentially expressed in male lesional and uninvolved skin and were not differentially expressed in female lesional and uninvolved skin compared to the skin of healthy volunteers.


*3.3. The Identified Estrogen-Responsive Genes Were Differentially Expressed in Menopausal and Non-Menopausal Patients, and Their Expression Was Influenced by the Disease*

Due to the small sample size of the performed LC–MS/MS study, we confirmed the differential expression of the identified estrogen-responsive proteins (ERPs) in women using qPCR and ELISA. The analysis of gene expression in skin samples by qPCR revealed that the genes encoding the identified ERPs were differentially expressed in lesional skin of qPCR/ELISA patients (n = 20) compared to the skin of qPCR/ELISA healthy volunteers (n = 11). Five identified genes, namely *HMOX1* (38.97 ± 4.91; *p* = 1.30 × 10−<sup>7</sup> ), *KRT19* (45.90 ± 5.86; *p* = 1.52 × 10−<sup>7</sup> ), *LDHA* (7.30 ± 2.55; *p* = 0.01), *HSPD1* (17.32 ± 3.57; *p* = 1.07 × 10−<sup>4</sup> ), and *MAPK1* (3.20 ± 0.77; *p* = 0.01), were induced, whereas *CA2* (0.43 ± 0.13; *p* = 0.01) was suppressed in lesional skin (Figure 3).

The expression profiles in qPCR/ELISA patients with and without menopause (Figure 3) were the same, i.e., the genes upregulated in patients without menopause (n = 10) were also upregulated in patients with menopause (n = 10) and vice versa, compared to qPCR/ELISA healthy volunteers (n = 11). All six identified ERGs had higher expression in patients without menopause compared to patients with menopause. Moreover, when patients with and without menopause were compared to each other, the changes in the expression of four genes, namely *HMOX1* (*p* = 0.001), *HSPD1* (*p* = 0.008), *CA2* (*p* = 0.006), and *MAPK1* (*p* = 0.012), were significant. In contrast, we did not see significant changes in gene expression (Figure 3) when we compared qPCR/ELISA healthy volunteers with and without menopause (n = 6 and 5, respectively).

The comparison of gene expression in qPCR/ELISA patients and healthy volunteers without menopause (n = 10 and 6, respectively) revealed a differential expression of five genes, namely *HMOX1* (*p* = 1.13 × 10−<sup>6</sup> ), *KRT19* (*p* = 1.81 × 10−<sup>4</sup> ), *HSPD1* (*p* = 9.00 × 10−<sup>4</sup> ), *LDHA* (*p* = 0.047), and *MAPK1* (*p* = 0.004), as depicted in Figure 3. *HMOX1*, *KRT19*, *HSPD1*, and *MAPK1* were upregulated in patients compared to healthy volunteers, whereas *CA2* was downregulated. Considering the fact that the compared samples belonged to the individuals without menopause, we suggested that the observed changes in gene expression were caused by the disease. Similar results were obtained when we compared qPCR/ELISA patients and healthy volunteers with menopause (n = 10 and 5, respectively). In patients, changes in the expression of *HMOX1*, *KRT19*, *LDHA*, *HSPD1*, and *CA2* were significant (*p* < 0.05). *HMOX1*, *KRT19*, *LDHA*, and *HSPD1* were upregulated. The expression level of *MAPK1* did not change (*p* < 0.45) and *CA2* was downregulated.

In turn, the principle component analysis (PCA) of qPCR data revealed that a single factor (PC1) was responsible for 53% of the variability between the skin samples (Figure 3b). The K-mean clustering (Figure 3c) identified two clusters that contained samples of qPCR/ELISA psoriasis patients (n = 20) and healthy volunteers (n = 11). However, we could not completely separate patients with and without menopause, as well as similar groups of healthy volunteers.

A comparative analysis of gene expression in the PBMC obtained from the individuals that participated in the qPCR/ELISA experiments revealed significant changes in the expression of four genes, namely *HMOX1*, *HSPD1*, *LDHA*, and *KRT19*, whereas the expression *MAPK1* and *CA2* was not detected (Figure 3d). Similarly to skin cells, the expression levels of *HMOX1*, *HSPD1*, *LDHA*, and *KRT19* were higher in non-menopausal patients compared to menopausal patients. However, the changes in gene expression were statistically insignificant, except for *LDHA* (*p* = 0.047). Moreover, we did not see any significant changes in gene expression when we compared non-menopausal and menopausal volunteers.

In non-menopausal patients, the expression levels of *HMOX1*, *HSPD1*, *LDHA*, and *KRT19* were higher compared to non-menopausal volunteers (Figure 3d). In particular, we found that changes in the expression levels of *KRT19* and *HMOX1* were significant (*p* = 0.022 and 0.019, respectively), whereas changes in the expression levels of *HSPD1* and *LDHA* were statistically insignificant (*p* = 0.079 and 0.072, respectively). Similarly, the expression levels of *HMOX1*, *HSPD1*, *LDHA*, and *KRT19* were higher in menopausal patients compared to menopausal volunteers (Figure 3d). However, the changes in their expression levels were statistically insignificant (*p* = 0.0503 for *HMOX1*, *p* = 0.053 for *HSPD1*, *p* = 0.102 for *LDHA*, and *p* = 0.080 for *KRT19*).

' **Figure 3.** The expression of identified estrogen-responsive genes in clinical samples of psoriasis patients and healthy volunteers assessed by qPCR. (**A**). The levels of gene expression in the samples of patients' lesional skin and in the skin of healthy volunteers. (**B**). Assessment of a variation in gene expression in the lesional skin of psoriasis patients and healthy volunteers by principle component analysis. (**C**). The plot of two first principal components (PC1 and PC2). Different groups are indicated by data points of different colors and shapes: group 1 is represented by blue diamonds; group 2 is represented by red squares; group 3 is represented by green triangles; and group 4 is represented by yellow circles. (**D**). The expression of identified estrogen-responsive genes in the PBMC obtained from the blood of patients and 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. Gene expression in menopausal patients was set equal to 1.
