Unique profile of antimicrobial peptide expression in polymorphic light eruption lesions compared to healthy skin, atopic dermatitis, and psoriasis

Summary Background Polymorphic light eruption (PLE) has been attributed to type IV, most likely delayed‐type hypersensitivity response (adaptive immunity) but little is known on innate immunity, especially antimicrobial peptides (AMPs) in the disease. Abnormalities in AMP expression have been linked to pathological skin conditions such as atopic dermatitis (AD) and psoriasis. Methods Antimicrobial peptide profiling was carried out in PLE skin samples (n,12) compared with that of healthy (n,13), atopic (n,6), and psoriatic skin (n,6). Results Compared to healthy skin, we observed increased expression of psoriasin and RNAse7 (both mostly in stratum granulosum of the epidermis), HBD‐2 (in the cellular infiltrate of the dermis), and LL37 (mostly in and around blood vessels and glands) in PLE lesional skin, a similar expression profile as present in psoriatic skin and different to that of AD (with little or no expression of psoriasin, RNAse7, HBD‐2, and LL37). HBD‐3 was downregulated in PLE compared to its high expression in the epidermis and dermis of healthy skin, AD, and psoriasis. Conclusion The unique profile of differentially expressed AMPs in PLE implies a role in the pathophysiology of the disease, possibly directly or indirectly linked to the microbiome of the skin.

newly formed (photo) antigens 12 probably due to regulatory T cells, 14,15 whereas in PLE prone subjects, a failure of immune suppression might favor the occurrence of the skin rash of the disease.
Skin cells produce small 10-50 amino acid residues known as antimicrobial peptides (AMPs) which have the potential to neutralize invading microorganisms. 16 AMPs can be classified into defensins (α-and β-defensins) and cathelicidin (LL37), 17 and other AMPs such as ribonuclease 7 (RNase7), psoriasin (S100A7), and dermcidin (sweat gland derived). 18 AMPs also take part in activating and mediating adaptive immune response. [19][20][21][22][23] Dysregulation in AMP production has been linked to many pathological skin conditions such as psoriasis, atopic dermatitis (AD), rosacea, and others. 18 We hypothesize that there might be an overall altered expression of AMPs as well in PLE lesions induced directly by UVR or due to possible UV-induced damage to certain microbes or microbial elements within or on the surface of the skin and such events could contribute to the pathogenesis of the disease. 12,24 We herein investigate the expression of psoriasin, RNase7, HBD-2, HBD-3, and LL-37, as these are the most commonly studied AMPs linked to many disease pathologies, using immunohistochemical stainings of lesional skin of PLE and compare it with that of healthy skin, lesional skin of AD and psoriasis patients.    used for statistical analysis. For RNase7 and HBD-3, we quantified expression using ImmunoRatio plugin 25 in ImageJ software with the images acquired with the Olympus DP71 digital camera. The percentage of the DAB-stained nuclear area over the total nuclear area in epidermis and dermis was calculated, and percentages were subjected to statistical analysis.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 6. For immunohistochemistry score comparison, unpaired nonparametric, Kruskal-Wallis test was used; each P-value was adjusted to account for multiple comparisons. A P-value smaller than .05 has been set as statistically significant.

| RESULTS
Psoriasin was the most highly expressed by keratinocytes in PLE and psoriasis ( Figure  In healthy skin ( Figure 1A), expression of psoriasin was found in stratum granulosum but was less intense and patchy compared to PLE or psoriasis. Overall, the expression of psoriasin in PLE was statistically significantly higher compared to healthy skin ( Figure 2A). Overall, the increased expression of LL-37 in PLE and psoriasis was significant compared to healthy skin or AD ( Figure 2E).
Statistical correlation analysis revealed the expression of AMPs was independent of age, except for psoriasin in PLE (r = .7012, P = .0095).
Moreover, there was no overall statistical correlation between sex and expression of any of the AMPs of this study.

| DISCUSSION
AMPs are produced in the skin during inflammation and/or in cases of infection. It has been demonstrated that UVR can induce production of keratinocyte-derived AMPs both in vitro and in vivo in humans and rodents. [26][27][28][29][30] Previous work on normal human keratinocytes revealed a dose-dependent increase in human β-defensin-2, β-defensin-3, RNase7, and psoriasin (S100A7) after UVB radiation. Note should be taken that interindividual variations are observed in expression of AMPs by UVR. 27,29,31 We now report a unique profile of protein expression of psoriasin, RNase7, HBD-2, HBD-3, and LL-37 in PLE occurring after natural sunlight exposure.
Psoriasin is reported to be expressed in inflamed skin and can exert a chemotactic influence on inflammatory cells. 32 In our study, psoriasin was highly expressed in PLE lesions and psoriatic skin, compared to healthy skin or AD (for the latter two diseases consistent with previous reports [33][34][35][36] ). However, we did not detect psoriasin expression in AD; indeed, the data on it in AD are controversial, possibly due to differences in analytical methodologies employed. 37  , the number of psoriasin-positive cells in the epidermis was significantly higher than in healthy skin but slightly lower compared to that in psoriasis. (B) Expression of RNase7 in PLE was significantly higher than compared to that of healthy skin, atopic dermatitis (AD) or psoriasis. (C) HBD-2 was significantly increased in PLE and psoriasis and to a lower degree increased in AD compared to healthy skin. (D) The expression of HBD-3 was (significantly) decreased in PLE compared to AD, psoriasis, and healthy skin. (E) Similar to psoriasis, PLE showed significantly increased expression of LL37 compared to healthy skin and AD. Data presented as mean with SEM psoriasin expression. [38][39][40][41] In PLE, IL-1β levels have been found to be elevated, 42 and this could induce high psoriasin expression in the skin.

Psoriasin displays a strong, selective antimicrobial activity against
Escherichia coli strains, 40,43 and this could theoretically hint toward the involvement of E. coli or its components in PLE lesions which is yet to be investigated. 12 Ribonuclease 7 is produced vastly by keratinocytes. It shows a strong bactericidal activity against a broad-spectrum of Gramnegative and Gram-positive bacteria. 44,45 We found that RNase7 was more strongly expressed in lesional PLE skin than in healthy skin and AD or psoriatic skin. RNase7 is also constitutively expressed in healthy skin, 46 shows also immunomodulatory functions on Th2 cells and cytokine production. 53 Human β-defensin 2 was first isolated from extracts obtained from psoriatic skin. 54 It is known to be upregulated in the skin by LPS, TNFα, IL-1β, IL-1α and bacterial infections 55 and also 1,25-dihydroxyvitamin D 3. 56 We found HBD-2 to be highly expressed in PLE, especially in infiltrating cells in the dermis. In this regard, it is already known in PLE that there is a lack of neutrophils and TNFα, 57 but an increase in IL-1β 58 production which could induce the expression of HBD-2. 42,59 In ex vivo experimental setting, the proinflammatory mediators TNFα and IL-17 but not UVR stimulated the expression of HBD-2. 60 However, in vivo data showed more heterogeneous expression for HBD-2 by UVR. 27 The presence of LPS and other microbial products in PLE remains to be determined. Lande et al 61 found that HBD-2 and HBD-3 have strong ability to break the tolerance to human DNA and form complexes with nucleic acids and trigger an innate immune response. It is known that the UVB waveband of sunlight can modify nucleic acids which could then become potential antigens, 5,12,62 provoking an immune reaction as seen in PLE, linked to higher expression of HBD-2 possibly exaggerating inflammation.
HBD-3 is expressed by the same cells as HBD-2 in the skin. We found that the expression of HBD-3 in PLE lesions was considerably lower than in healthy skin, and lesional skin of AD and psoriasis patients. The inducers for upregulating HBD-3 are similar to those of HBD-2. Among other β-defensins, HBD-3 is the only AMP which is regulated by insulin-like growth factors such as (IGF-1) and transforming growth factor (TGFα) and microbial stimuli (such as LPS, peptidoglycan, or SpeB [a virulence factor from Streptococcus pyogenes]) to keratinocytes in vitro. 63 Although UVR is known to induce expression of HBD-3, our observations of impaired expression in PLE lesions might imply its functional role in disease pathogenesis as HBD-3 is known to be involved with keratinocyte proliferation and migration by activating EGFR, STAT1, and STAT3. 21 HBD-3 is known to be more potent in killing microbes than other defensins. 64 The lower or reduced expression of HBD-3 in PLE lesions in our study and a previous report 26 could suggest a differential microbial landscape in PLE patients that fails to induce HBD-3, similarly as observed in AD where lower HBD-3 expression corresponds to higher Staphylococcus aureus colonization. 65,66 LL-37 is the only cathelicidin peptide detected in humans and produced in much vast quantity in psoriasis and is known to play a major role in the inflammatory cascade driving psoriatic disease. [67][68][69][70] Herein and in a previous report, 71  forming aggregates with self-nucleic acids. [68][69][70] However, in PLE patients a complete absence of pDCs has been reported, 77 suggesting that the high expression of LL-37 might be involved in other inflammatory processes. In psoriasis patients, LL-37 has been recognized as an autoantigen that stimulates circulating T cells and contributes to the autoimmunity in these patients. 78 As a similar autoimmune environment may obviously exist in PLE patients, it is likely that there are increased LL-37-specific T cells. On the other hand, LL-37 shows broad antimicrobial activity to various microbes and the high expression of this peptide could be directly involved in the antimicrobial activity in PLE patients.
The major limitations of this study were the limited sample size and the imperfect matching in age and sex of the different patient groups. However, our analysis showed that the expression of AMPs (except for psoriasin that was age-related in PLE) was neither age-nor sex-dependent in the sample sets of the study. Indeed, age-matched sample (data not shown) analysis revealed a similar picture than the overall analysis shown in Figure 2. Furthermore, previous work has shown that psoriasin, RNase7, and HBD-3 was not only upregulated in UVR-provoked lesional PLE skin but also to a weaker extent in UVR-exposed skin of PLE patients without eruption. However, as our study lacks normal skin from PLE patients, we cannot conclude on the magnitude of AMP expression by UVR without PLE eruption and if UVR increases AMP expression in PLE lesions above levels of nonlesional skin of PLE patients not mounting PLE after UV exposure.
Hence, the results of this study must be interpreted in the light of the study's limitations. Previous research has shown consistently with our results a certain extent of altered expression levels of AMPs (psoriasin, RNase7, HBD-2, HBD-3) in a limited number of PLE lesions photoprovoked by artificial UV-A radiation. 26 Our findings of the unique expression pattern of AMPs, including LL-37 (that at least hypothetically could be a potential driver in PLE) provide further understanding of the pathogenesis of the disease and could help unraveling a complex network between AMPs, microbiome, and immune system. That said, the potential case of altered microbial landscape in PLE patients is yet to be investigated, but the altered expression of various AMPs among different skin conditions in our study strongly implies that either microbial elements or microbes themselves may be involved in the pathogenesis of PLE. Indeed, these microbial elements could be the source of the yet undetected antigens formed in PLE patients after exposure to UVR. 12