mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Expression of antimicrobial peptides and toll-like receptors is increased in tinea and pityriasis versicolor € rig, B. Neumann and E. Proksch J. Brasch, A. Mo Department of Dermatology, University Hospitals of Schleswig-Holstein, Kiel, Germany
Summary
In superficial tinea and pityriasis versicolor, the causative fungi are for the most part confined to the stratum corneum which is barely reached by leukocytes. Therefore, a role of non-cellular components in the epidermal antifungal defence was suggested. To investigate the presence of such factors in these infections, the expression of human beta defensins 2 and 3 (hBD-2, hBD-3), RNase 7, psoriasin, toll-like receptors 2, 4 and 9 (TLR2, TLR4 and TLR9) and dectin 2 was analysed by use of immunostainings in skin biopsies. We found that hBD2, hBD3, psoriasin, RNase7, TLR2 and TLR4 were significantly more often expressed in distinct layers of lesional epidermis as compared with uninfected epidermis. In both infections but not in normal skin, hBD2 and hBD3 were commonly expressed within the stratum corneum and in the stratum granulosum. Similarly, psoriasin was seen more often in the upper skin layers of both infections as compared with normal skin. No significant differences between normal and infected skin were found for the expression of TLR9 and dectin 2. Our findings clearly show the expression of specific antimicrobial proteins and defence-related ligands in superficial tinea as well as in pityriasis versicolor, suggesting that these factors contribute to fungal containment.
Key words: Dermatophytes, Malassezia, defensins, toll-like receptors, psoriasin, dectin.
Introduction Superficial tinea and pityriasis versicolor are both very common fungal skin infections occurring worldwide. In most cases, the causative dermatophytes and Malassezia spp. spread within the stratum corneum of the epidermis. During infection, a moderate inflammatory response is regularly observed. This response is characterised by a mild dermal infiltrate of mononuclear cells and polymorphous leucocytes. Although only few of these inflammatory cells reach the superficial skin layers, the skin invading fungi are normally confined to the stratum corneum. This suggests that the skin Correspondence: Prof. Dr. med. Jochen Brasch, Department of Dermatology, Venerology and Allergology, University Hospitals of SchleswigHolstein, Campus Kiel, Schittenhelmstr.7, 24105 Kiel, Germany. Tel.: +49 431 5971507. Fax: +49 431 5971611. E-mail: [email protected] Submitted for publication 14 June 2013 Accepted for publication 18 July 2013
© 2013 Blackwell Verlag GmbH
may harbour natural non-cellular components against fungi. The antimicrobial peptides human beta defensins 2 and 3 (hBD-2, hBD-3), RNase 7 and psoriasin are derived from keratinocytes of the epidermis and express antibacterial activity. Recent investigations have shown that some of these antimicrobial peptides may also be part of the natural defence against fungal infection.1–5 Toll-like receptors (TLRs) and dectins are an important part of the innate immunity. They recognise pathogen-associated molecular patterns, conserved motifs predominantly found in microorganisms and hence act as pattern recognition receptors.6,7To deepen the knowledge about the pathogenetic processes involved in these defence mechanisms, we have now investigated the expression of antimicrobial proteins derived from keratinocytes of the epidermis and respective signalling molecules involved in innate immunity within the lesional human epidermis of superficial tinea and pityriasis versicolor. Skin biopsies of such lesions were immunohistologically stained for constituents of the natural defence and the findings in
doi:10.1111/myc.12118
J. Brasch et al.
tinea, pityriasis versicolor and controls from normal skin were compared.
Material and methods Skin biopsies that in our hospital had been taken for diagnostic reasons from patients with superficial tinea and pityriasis versicolor were used for this study. In all biopsies, typical fungal elements had been microscopically verified within the stratum corneum by PAS stains. Seventeen tinea biopsies were used that all had been taken from typical lesions of superficial tinea corporis. In nine of these cases, Trichophyton rubrum had been identified as the causative agent by cultures, in the other eight cases the dermatophyte had not been identified. Ten biopsies of clinically typical truncal lesions of pityriasis versicolor with microscopically demonstrated characteristic fungal elements were included. No cultures had been performed in these cases. Ten control specimens of uninfected skin were obtained from the borders of skin excisions that had been done to remove benign melanocytic nevi from the trunk of otherwise healthy patients. All biopsies had been collected in the time from 2002 to 2008 and were fixed in formaldehyde and embedded in paraffin. For this study, separate histological slides were prepared from the embedded tissues. Slices with 3–5 lm thickness were cut with a microtome and used for immunostainings with an avidin-biotin method. In short, the tissue slices were placed on slides, cleaned from paraffin with xylene and rehydrated. To reduce background staining, a 3% peroxide solution was shortly applied. The specimens were then heated to 90 °C in citrate buffer for 30 min and after cooling off, they were washed with tris buffered saline (TBS). Thereafter, unspecific staining was blocked by incubation for 30 min with rabbit or pig serum. After removal of the
blocking serum antibodies to human beta defensin 2 (hBD-2), human beta defensin 3 (hBD-3), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), toll-like receptor 9 (TLR9), dectin 2, RNase 7 or psoriasin were added for 60 min. The specifications of the antibodies used are shown in Table 1. Then unbound antibodies were rinsed off and the specimens were exposed to an appropriate biotinylated secondary antibody for 30 min. Then they were washed again with TBS and overlaid with StreptABComplex (DAKO, Hamburg, Germany) for 30 min. After another washing with TBS, staining was completed by adding peroxidase substrate solution (BioGenex, Fremont, CA) according to the manufacturer’s instructions. The preparations were then washed with distilled water, exposed to tap water and covered with glycerol and cover slips. The staining results were evaluated by separately assessing the stratum corneum, stratum granulosum, stratum spinosum and stratum basale of each slide. For each tissue layer and target, the staining was graded as positive or negative by light microscopy. The results obtained with the distinct markers in the slides of tinea, pityriasis versicolor and normal skin were statistically analysed by the use of Fisher’s global exact test. For comparisons between normal skin and the fungal infections, P-values were adjusted for multiple tests according to Holm.8 A P-value < 0.05 was considered to indicate statistical significance.
Results We determined the expression of relevant defensins and signalling molecules in human fungal diseases by immunostaining. There was no significant difference between superficial tinea caused by T. rubrum and tinea caused by unidentified dermatophytes. Therefore, the data of all tinea lesions were pooled for the
Table 1 Antibodies.
Target
Source
Clonality
Dilution (in tris buffered saline)
Human beta defensin 2 (hBD2) Human beta defensin 3 (hBD3) Toll-like receptor 2 (TLR2) Toll-like receptor 4 (TLR4) Toll-like receptor 9 (TLR9) Dectin 2 RNAse 7
Goat Rabbit Rabbit Rabbit Goat Rabbit Goat
Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal
1 1 1 1 1 1 1
Psoriasin
Mouse
Monoclonal
1 : 20000
2
: : : : : : :
500 500 500 100 250 75 400
Supplier Preprotech, Hamburg, Germany Preprotech, Hamburg, Germany Abcam, Cambridge, UK Abcam, Cambridge, UK Abcam, Cambridge, UK Novus biologicals, Littleton, CO, USA €rgen Harder, Department of Dermatology, Prof. Dr. Ju UKSH, Kiel, Germany Dr. Hans Lange, Institute for Biochemistry, CAU Kiel
© 2013 Blackwell Verlag GmbH
AMPs and TLRs in superficial mycoses
following analyses. In general, pronounced staining for all the defensins and the signalling molecules was noticed more often in specific skin layers of mycotic lesions as compared with normal skin. Table 2 in detail shows the percentages of all specimens with positive staining for each distinct marker in the different skin layers for tinea, pityriasis versicolor and normal skin. hBD2 was commonly expressed in stratum corneum of both infections and also in stratum granulosum of tinea. A typical example is shown Table 2 Percentages of specimen with positive staining for the targets shown in column 1 within the indicated epidermal layers in tinea, pityriasis versicolor and normal skin.
in Fig. 1. hBD2 was not seen in normal skin. hBD3 was also significantly more often expressed in the stratum corneum of tinea and pityriasis versicolor as compared with uninfected epidermis, which expressed hBD3 in the stratum corneum in 40% of the biopsies. Psoriasin was expressed more often in the stratum corneum, in the stratum granulosum and in the stratum spinosum of tinea (Fig. 2). The pattern of psoriasin expression in pityriasis versicolor was similar to the corresponding one in tinea.
Pityriasis versicolor
Tinea hBD-2 Stratum Stratum Stratum Stratum hBD-3 Stratum Stratum Stratum Stratum TLR2 Stratum Stratum Stratum Stratum TLR4 Stratum Stratum Stratum Stratum TLR9 Stratum Stratum Stratum Stratum Dectin2 Stratum Stratum Stratum Stratum RNase7 Stratum Stratum Stratum Stratum Psoriasin Stratum Stratum Stratum Stratum
Normal skin
corneum granulosum spinosum basale
82.4* (59–93.8) 82.4* (59–93.8) 47.1 (26.2–60) 0 (0–18.5)
100* (72.2–100) 30 (10.8–60.3) 0 (0–27.8) 0 (0–27.8)
0 0 0 0
corneum granulosum spinosum basale
94.1* (73–99) 41.2 (21.6-64) 0 (0–25.8) 0 (0–18.4)
100* (72.2–100) 20 (5.7–51) 0 (0–25.8) 0 (0–27.8)
40 0 0 0
corneum granulosum spinosum basale
10.5* (2.9–31.4) 21.1 (8.5–42.4) 73.7** (51.2–88.2) 73.7** (51.2–88.2)
0* (0–27.8) 10 (1.8–40.4) 10 (1.8–40.4) 10 (1.8–40.4)
83.3 (55.2–95.3) 25 (8.9–53.2) 41.7 (19.3–68) 41.7 (19.3–68)
corneum granulosum spinosum basale
52.9** (30.9–73.8) 70.6 (48.9–86.7) 82.4 (58.6–93.6) 70.6 (46.9–86.7)
0 50 60 30
(0–27.8) (23.7–76.3) (31.3–83.1) (10.8–60.3)
45.5 36.4 36.4 36.4
(21.3–72) (15.2–64.7) (15.2–64.7) (15.2–64.7)
corneum granulosum spinosum basale
17.6** (6.2–41) 35.3 (17.3–58.7) 70.6 (46.9–86.7) 29.4 (13.3–53.1)
100 30 20 0
(72.2–100) (10.8–60.3) (5.7–51) (0–27.8)
66.7 33.3 41.7 33.3
(39.1–86.2) (13.8–60.9) (19.3–68) (13.8–60.9)
corneum granulosum spinosum basale
88.9 72.2 83.3 44.4
90 60 50 40
(59.6–98.2) (31.3–83.1) (23.7–76.3) (16.8–68.7)
90 60 60 60
(72.2–100) (23.7–76.3) (0–27.8) (0–27.8)
92.9 (68.6–98.7) 21.4 (7.6–47.6) 0 (0–21.5) 0 (0–21.5)
(67.2–96.9) (54.2–90.6) (60.7–94.1) (24.5–66.2)
corneum granulosum spinosum basale
100 (81.6–100) 94.1* (73–99) 0 (0–18.4) 0 (0–18.4)
100 50 0 0
corneum granulosum spinosum basale
88.2* (65.6–96.7) 100* (81.6–100) 76.5* (52.8–90.5) 0 (0–18.4)
100* (72.2–100) 90* (59.6–98.2) 80* (49–94.3) 10 (1.6–40.4)
(0–35.4) (0–35.4) (0–35.4) (0–35.4) (16.8–68.7) (0–27.8) (0–25.8) (0–27.8)
(16.8–99.8) (31.3–83.2) (31.3–83.2) (31.3–83.2)
36.4 (15.2–64.7) 0 (0–25.9) 0 (0–25.9) 0 (0–25.9)
The 95% interval of confidence is shown in brackets. Statistically significant differences (P < 0.05) between tinea and normal skin and between pityriasis versicolor and normal skin are indicated by *in the column of the disease, statistically significant differences between tinea and pityriasis versicolor are indicated by **in the column of tinea.
© 2013 Blackwell Verlag GmbH
3
J. Brasch et al.
Fig. 1 Positive staining for hBD2 in superficial epidermal layers
Fig. 3 Positive staining for RNAse 7 in stratum corneum and
of tinea.
granulosum of tinea.
Fig. 2 Positive staining for psoriasin in stratum granulosum and
Fig. 4 Positive staining for TLR2 in stratum spinosum of tinea.
upper stratum spinosum of tinea.
RNAse 7 was regularly detected in the stratum corneum of tinea pityriasis, versicolor and normal skin. However, RNAse 7 staining was significantly more often positive in the stratum granulosum of tinea (Fig. 3) than in the stratum granulosum of pityriasis versicolor and normal skin. TLR2 and TLR4 were more often detected in infected skin of tinea and pityriasis versicolor than in normal epidermis. A typical example for positive TLR 2 staining in the stratum spinosum of tinea is shown in Fig. 4. In contrast to the defensins, these TLRs were prevalently found in the lower epidermal layers. In pityriasis versicolor, staining for TLR2 and TLR4 was even absent in the stratum corneum. TLR9 was also mostly found in the stratum spinosum of tinea and in
4
the stratum corneum of pityriasis versicolor, but was detected in the stratum corneum of normal skin as well. Dectin 2 was detected in all layers of the epidermis in both fungal infections and in normal epidermis, too, without significant differences.
Discussion We performed a retrospective study on the expression of antimicrobial peptides and signalling molecules in lesional skin of human tinea and pityriasis versicolor. Some limitations need to be considered. A correlation with clinical data was not possible and presumably the biopsies studied came from lesions with different degrees of inflammation and had been taken at different phases of their development. Knowing this, no
© 2013 Blackwell Verlag GmbH
AMPs and TLRs in superficial mycoses
attempt was made to differentiate between early and acute lesions or between chronic and regressive ones. Furthermore, due to the fact that the biopsies were probably obtained during diverse clinical situations, a considerable heterogeneity of results was to be expected. Because a trustworthy grading of the intensity of immunostaining is not available, we decided to only discriminate between positive and negative staining. This is certainly not a very sensitive method, but in our opinion is adequate to the conditions, avoiding any pretence of an exactness that could not be ensured. Despite these restrictions, our findings clearly show that in superficial tinea as well as in pityriasis versicolor, an expression of defence-related antimicrobial peptides and of signalling molecules occurs. Our findings are in agreement with published data in infections with Candida and other fungi.9–14 The localization of the antimicrobial proteins within the upper epidermis also suggests a role in the defence against pathogens invading the skin from the outside. In view of the superficial localization of the agents, it is not surprising that the expression of these factors is mostly found in the upper epidermal layers. The antimicrobial peptides hBD2, hBD3 and psoriasin were scarcely detected in normal skin, but clearly upregulated in fungal infections. Interestingly, an occasional expression of hBD3 and psoriasin was confined to the stratum corneum only in normal skin, whereas in infected skin the staining extended to the stratum granulosum and stratum spinosum. Also, RNase7 was seen more often in the stratum granulosum of tinea compared with normal skin. The shift of the antimicrobial peptide expression to deeper layers of the epidermis was more pronounced in tinea compared with pityriasis versicolor, suggesting a correlation with the higher invasiveness of tinea. TLR2 and TLR4 can be expressed in normal skin, but are clearly more pronounced in fungal infection. In addition, a shift in the expression of TLR2 and TLR4 was noticed from the upper layers in normal skin to the lower epidermal layers in fungal infection. Again, the effects were more pronounced in tinea compared with pityriasis versicolor. This suggests that in severely infected skin, the innate immune defence already starts in the lower epidermis. We found no significant differences in the expression of TLR9 and dectin 2 in infected compared with normal skin. We conclude that the expression of TLR2 and TLR4 is very likely stimulated by the fungi, whereas a role of TLR9 and dectin 2 is uncertain.
© 2013 Blackwell Verlag GmbH
In view of the superficial localization of the pathogens, it is not surprising that the expression of the investigated factors is mostly found in the upper epidermal layers. The antimicrobial factors hBD2, hBD3 and psoriasin are clearly upregulated in the superficial fungal infections and they are mainly localised within the upper epidermis. It thus appears reasonable to hypothesise that these antimicrobial peptides and signalling molecules are involved in a stimulated defence reaction of the epidermis induced by the invaded fungi. Several factors of this system can have antifungal effects under certain conditions.5,15–18 We conclude from our findings that the innate immune system is activated in the process of superficial tinea and pityriasis versicolor by the causative fungi. Although in these infections, the fungi are mainly localised within the non-vital stratum corneum, it occurs that even in this remote ecological niche they are not out of reach for an antimicrobial defence mounted by living keratinocytes of the epidermis.
Conflicts of interest None
References Sawamura D, Goto M, Shibaki A et al. Beta defensin-3 engineered epidermis shows highly protective effect for bacterial infection. Gene Ther 2005; 12: 857–61. 2 De Jongh GJ, Zeeuwen PLJM, Kucharekova M et al. High expression levels of keratinocyte antimicrobial proteins in psoriasis compared with atopic dermatitis. J Invest Dermatol 2005; 125: 1163– 73. 3 Zanger P, Holzer J, Schleucher R, Steffen H, Schittek B, Gabrysch S. Constitutive expression of the antimicrobial peptide RNase 7 is associated with Staphylococcus aureus infection of the skin. J Infect Dis 2009; 200: 1907–15. 4 Zheng Y, Niyonsaba F, Ushio H et al. Microbicidal protein psoriasin is a multifunctional modulator of neutrophil activation. Immunology 2008; 124: 357–67. 5 Akira S. Toll-like receptor signaling. J Biol Chem 2003; 278: 38105–8. 6 Saijo S, Iwakura Y. Dectin-1 and Dectin-2 in innate immunity against fungi. Int Immunol 2011; 23: 467–72. 7 Gl€ aser R, Meyer-Hoffert U, Harder J et al. The antimicrobial protein psoriasin (S100A7) is upregulated in atopic dermatitis and after experimental skin barrier disruption. J Invest Dermatol 2009; 129: 641–9. 8 Holm S. A simple sequentially rejective multiple test procedure. Scand J Statistics 1979; 6: 65–70. 9 Curry JL, Qin J-Z, Bonish B et al. Innate immune-related receptors in normal and psoriatic skin. Arch Pathol Lab Med 2003; 127: 178– 86. 10 Kiemer AK. Angeborenes Immunsystem. TLR ero¨ffnen neue Mo¨glichkeiten. Pharmazeutische Zeitung. http://www.pharmazeutische-zeitung.de/index.php?id=4546(online 04/2008). 11 Bauer S, Kirschning CJ, H€ acker H et al. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA 2001; 98: 9237–42. 1
5
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12
13
14
6
Criado PR, de Oliveira CB, Dantas KC, Takiguti FA, Benini LV, Vasconcellos C. Superficial mycosis and the immune response elements. An Bras Dermatol 2011; 86: 726–31. Gambichler T, Skrygan M, Tomi NS et al. Differential mRNA expression of antimicrobial peptides and proteins in atopic dermatitis as compared to psoriasis vulgaris and healthy skin. Int Arch Allergy Immunol 2008; 147: 17–24. Proksch E, Brasch J. Barrier function, epidermal differentiation, and human beta- defensin 2 expression in tinea corporis. J Invest Dermatol 2007; 127: 1720–7.
15
16 17 18
Gl€ aser R, K€ oten B, Wittersheim M, Harder J. Psoriasin: key molecule of the cutaneous barrier? J Dtsch Dermatol Ges. http://www.ncbi.nlm. nih.gov/pubmed/21501383 Schr€ oder J-M, Harder J. Human beta-defensin-2. Int J Biochem Cell Biol 1999; 31: 645–51. Schr€ oder JM, Harder J. Antimicrobial skin peptides and proteins. Cell Mol Life Sci 2006; 63: 469–86. Fritz P, Beck-Jendroschek V, Brasch J. Inhibition of dermatophytes by the antimicrobial peptides human b-defensin-2, ribonuclease 7 and psoriasin. Med Mycol 2012; 50: 579–84.
© 2013 Blackwell Verlag GmbH
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Expression of antimicrobial peptides and toll-like receptors is increased in tinea and pityriasis versicolor € rig, B. Neumann and E. Proksch J. Brasch, A. Mo Department of Dermatology, University Hospitals of Schleswig-Holstein, Kiel, Germany
Summary
In superficial tinea and pityriasis versicolor, the causative fungi are for the most part confined to the stratum corneum which is barely reached by leukocytes. Therefore, a role of non-cellular components in the epidermal antifungal defence was suggested. To investigate the presence of such factors in these infections, the expression of human beta defensins 2 and 3 (hBD-2, hBD-3), RNase 7, psoriasin, toll-like receptors 2, 4 and 9 (TLR2, TLR4 and TLR9) and dectin 2 was analysed by use of immunostainings in skin biopsies. We found that hBD2, hBD3, psoriasin, RNase7, TLR2 and TLR4 were significantly more often expressed in distinct layers of lesional epidermis as compared with uninfected epidermis. In both infections but not in normal skin, hBD2 and hBD3 were commonly expressed within the stratum corneum and in the stratum granulosum. Similarly, psoriasin was seen more often in the upper skin layers of both infections as compared with normal skin. No significant differences between normal and infected skin were found for the expression of TLR9 and dectin 2. Our findings clearly show the expression of specific antimicrobial proteins and defence-related ligands in superficial tinea as well as in pityriasis versicolor, suggesting that these factors contribute to fungal containment.
Key words: Dermatophytes, Malassezia, defensins, toll-like receptors, psoriasin, dectin.
Introduction Superficial tinea and pityriasis versicolor are both very common fungal skin infections occurring worldwide. In most cases, the causative dermatophytes and Malassezia spp. spread within the stratum corneum of the epidermis. During infection, a moderate inflammatory response is regularly observed. This response is characterised by a mild dermal infiltrate of mononuclear cells and polymorphous leucocytes. Although only few of these inflammatory cells reach the superficial skin layers, the skin invading fungi are normally confined to the stratum corneum. This suggests that the skin Correspondence: Prof. Dr. med. Jochen Brasch, Department of Dermatology, Venerology and Allergology, University Hospitals of SchleswigHolstein, Campus Kiel, Schittenhelmstr.7, 24105 Kiel, Germany. Tel.: +49 431 5971507. Fax: +49 431 5971611. E-mail: [email protected] Submitted for publication 14 June 2013 Accepted for publication 18 July 2013
© 2013 Blackwell Verlag GmbH
may harbour natural non-cellular components against fungi. The antimicrobial peptides human beta defensins 2 and 3 (hBD-2, hBD-3), RNase 7 and psoriasin are derived from keratinocytes of the epidermis and express antibacterial activity. Recent investigations have shown that some of these antimicrobial peptides may also be part of the natural defence against fungal infection.1–5 Toll-like receptors (TLRs) and dectins are an important part of the innate immunity. They recognise pathogen-associated molecular patterns, conserved motifs predominantly found in microorganisms and hence act as pattern recognition receptors.6,7To deepen the knowledge about the pathogenetic processes involved in these defence mechanisms, we have now investigated the expression of antimicrobial proteins derived from keratinocytes of the epidermis and respective signalling molecules involved in innate immunity within the lesional human epidermis of superficial tinea and pityriasis versicolor. Skin biopsies of such lesions were immunohistologically stained for constituents of the natural defence and the findings in
doi:10.1111/myc.12118
J. Brasch et al.
tinea, pityriasis versicolor and controls from normal skin were compared.
Material and methods Skin biopsies that in our hospital had been taken for diagnostic reasons from patients with superficial tinea and pityriasis versicolor were used for this study. In all biopsies, typical fungal elements had been microscopically verified within the stratum corneum by PAS stains. Seventeen tinea biopsies were used that all had been taken from typical lesions of superficial tinea corporis. In nine of these cases, Trichophyton rubrum had been identified as the causative agent by cultures, in the other eight cases the dermatophyte had not been identified. Ten biopsies of clinically typical truncal lesions of pityriasis versicolor with microscopically demonstrated characteristic fungal elements were included. No cultures had been performed in these cases. Ten control specimens of uninfected skin were obtained from the borders of skin excisions that had been done to remove benign melanocytic nevi from the trunk of otherwise healthy patients. All biopsies had been collected in the time from 2002 to 2008 and were fixed in formaldehyde and embedded in paraffin. For this study, separate histological slides were prepared from the embedded tissues. Slices with 3–5 lm thickness were cut with a microtome and used for immunostainings with an avidin-biotin method. In short, the tissue slices were placed on slides, cleaned from paraffin with xylene and rehydrated. To reduce background staining, a 3% peroxide solution was shortly applied. The specimens were then heated to 90 °C in citrate buffer for 30 min and after cooling off, they were washed with tris buffered saline (TBS). Thereafter, unspecific staining was blocked by incubation for 30 min with rabbit or pig serum. After removal of the
blocking serum antibodies to human beta defensin 2 (hBD-2), human beta defensin 3 (hBD-3), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), toll-like receptor 9 (TLR9), dectin 2, RNase 7 or psoriasin were added for 60 min. The specifications of the antibodies used are shown in Table 1. Then unbound antibodies were rinsed off and the specimens were exposed to an appropriate biotinylated secondary antibody for 30 min. Then they were washed again with TBS and overlaid with StreptABComplex (DAKO, Hamburg, Germany) for 30 min. After another washing with TBS, staining was completed by adding peroxidase substrate solution (BioGenex, Fremont, CA) according to the manufacturer’s instructions. The preparations were then washed with distilled water, exposed to tap water and covered with glycerol and cover slips. The staining results were evaluated by separately assessing the stratum corneum, stratum granulosum, stratum spinosum and stratum basale of each slide. For each tissue layer and target, the staining was graded as positive or negative by light microscopy. The results obtained with the distinct markers in the slides of tinea, pityriasis versicolor and normal skin were statistically analysed by the use of Fisher’s global exact test. For comparisons between normal skin and the fungal infections, P-values were adjusted for multiple tests according to Holm.8 A P-value < 0.05 was considered to indicate statistical significance.
Results We determined the expression of relevant defensins and signalling molecules in human fungal diseases by immunostaining. There was no significant difference between superficial tinea caused by T. rubrum and tinea caused by unidentified dermatophytes. Therefore, the data of all tinea lesions were pooled for the
Table 1 Antibodies.
Target
Source
Clonality
Dilution (in tris buffered saline)
Human beta defensin 2 (hBD2) Human beta defensin 3 (hBD3) Toll-like receptor 2 (TLR2) Toll-like receptor 4 (TLR4) Toll-like receptor 9 (TLR9) Dectin 2 RNAse 7
Goat Rabbit Rabbit Rabbit Goat Rabbit Goat
Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal
1 1 1 1 1 1 1
Psoriasin
Mouse
Monoclonal
1 : 20000
2
: : : : : : :
500 500 500 100 250 75 400
Supplier Preprotech, Hamburg, Germany Preprotech, Hamburg, Germany Abcam, Cambridge, UK Abcam, Cambridge, UK Abcam, Cambridge, UK Novus biologicals, Littleton, CO, USA €rgen Harder, Department of Dermatology, Prof. Dr. Ju UKSH, Kiel, Germany Dr. Hans Lange, Institute for Biochemistry, CAU Kiel
© 2013 Blackwell Verlag GmbH
AMPs and TLRs in superficial mycoses
following analyses. In general, pronounced staining for all the defensins and the signalling molecules was noticed more often in specific skin layers of mycotic lesions as compared with normal skin. Table 2 in detail shows the percentages of all specimens with positive staining for each distinct marker in the different skin layers for tinea, pityriasis versicolor and normal skin. hBD2 was commonly expressed in stratum corneum of both infections and also in stratum granulosum of tinea. A typical example is shown Table 2 Percentages of specimen with positive staining for the targets shown in column 1 within the indicated epidermal layers in tinea, pityriasis versicolor and normal skin.
in Fig. 1. hBD2 was not seen in normal skin. hBD3 was also significantly more often expressed in the stratum corneum of tinea and pityriasis versicolor as compared with uninfected epidermis, which expressed hBD3 in the stratum corneum in 40% of the biopsies. Psoriasin was expressed more often in the stratum corneum, in the stratum granulosum and in the stratum spinosum of tinea (Fig. 2). The pattern of psoriasin expression in pityriasis versicolor was similar to the corresponding one in tinea.
Pityriasis versicolor
Tinea hBD-2 Stratum Stratum Stratum Stratum hBD-3 Stratum Stratum Stratum Stratum TLR2 Stratum Stratum Stratum Stratum TLR4 Stratum Stratum Stratum Stratum TLR9 Stratum Stratum Stratum Stratum Dectin2 Stratum Stratum Stratum Stratum RNase7 Stratum Stratum Stratum Stratum Psoriasin Stratum Stratum Stratum Stratum
Normal skin
corneum granulosum spinosum basale
82.4* (59–93.8) 82.4* (59–93.8) 47.1 (26.2–60) 0 (0–18.5)
100* (72.2–100) 30 (10.8–60.3) 0 (0–27.8) 0 (0–27.8)
0 0 0 0
corneum granulosum spinosum basale
94.1* (73–99) 41.2 (21.6-64) 0 (0–25.8) 0 (0–18.4)
100* (72.2–100) 20 (5.7–51) 0 (0–25.8) 0 (0–27.8)
40 0 0 0
corneum granulosum spinosum basale
10.5* (2.9–31.4) 21.1 (8.5–42.4) 73.7** (51.2–88.2) 73.7** (51.2–88.2)
0* (0–27.8) 10 (1.8–40.4) 10 (1.8–40.4) 10 (1.8–40.4)
83.3 (55.2–95.3) 25 (8.9–53.2) 41.7 (19.3–68) 41.7 (19.3–68)
corneum granulosum spinosum basale
52.9** (30.9–73.8) 70.6 (48.9–86.7) 82.4 (58.6–93.6) 70.6 (46.9–86.7)
0 50 60 30
(0–27.8) (23.7–76.3) (31.3–83.1) (10.8–60.3)
45.5 36.4 36.4 36.4
(21.3–72) (15.2–64.7) (15.2–64.7) (15.2–64.7)
corneum granulosum spinosum basale
17.6** (6.2–41) 35.3 (17.3–58.7) 70.6 (46.9–86.7) 29.4 (13.3–53.1)
100 30 20 0
(72.2–100) (10.8–60.3) (5.7–51) (0–27.8)
66.7 33.3 41.7 33.3
(39.1–86.2) (13.8–60.9) (19.3–68) (13.8–60.9)
corneum granulosum spinosum basale
88.9 72.2 83.3 44.4
90 60 50 40
(59.6–98.2) (31.3–83.1) (23.7–76.3) (16.8–68.7)
90 60 60 60
(72.2–100) (23.7–76.3) (0–27.8) (0–27.8)
92.9 (68.6–98.7) 21.4 (7.6–47.6) 0 (0–21.5) 0 (0–21.5)
(67.2–96.9) (54.2–90.6) (60.7–94.1) (24.5–66.2)
corneum granulosum spinosum basale
100 (81.6–100) 94.1* (73–99) 0 (0–18.4) 0 (0–18.4)
100 50 0 0
corneum granulosum spinosum basale
88.2* (65.6–96.7) 100* (81.6–100) 76.5* (52.8–90.5) 0 (0–18.4)
100* (72.2–100) 90* (59.6–98.2) 80* (49–94.3) 10 (1.6–40.4)
(0–35.4) (0–35.4) (0–35.4) (0–35.4) (16.8–68.7) (0–27.8) (0–25.8) (0–27.8)
(16.8–99.8) (31.3–83.2) (31.3–83.2) (31.3–83.2)
36.4 (15.2–64.7) 0 (0–25.9) 0 (0–25.9) 0 (0–25.9)
The 95% interval of confidence is shown in brackets. Statistically significant differences (P < 0.05) between tinea and normal skin and between pityriasis versicolor and normal skin are indicated by *in the column of the disease, statistically significant differences between tinea and pityriasis versicolor are indicated by **in the column of tinea.
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J. Brasch et al.
Fig. 1 Positive staining for hBD2 in superficial epidermal layers
Fig. 3 Positive staining for RNAse 7 in stratum corneum and
of tinea.
granulosum of tinea.
Fig. 2 Positive staining for psoriasin in stratum granulosum and
Fig. 4 Positive staining for TLR2 in stratum spinosum of tinea.
upper stratum spinosum of tinea.
RNAse 7 was regularly detected in the stratum corneum of tinea pityriasis, versicolor and normal skin. However, RNAse 7 staining was significantly more often positive in the stratum granulosum of tinea (Fig. 3) than in the stratum granulosum of pityriasis versicolor and normal skin. TLR2 and TLR4 were more often detected in infected skin of tinea and pityriasis versicolor than in normal epidermis. A typical example for positive TLR 2 staining in the stratum spinosum of tinea is shown in Fig. 4. In contrast to the defensins, these TLRs were prevalently found in the lower epidermal layers. In pityriasis versicolor, staining for TLR2 and TLR4 was even absent in the stratum corneum. TLR9 was also mostly found in the stratum spinosum of tinea and in
4
the stratum corneum of pityriasis versicolor, but was detected in the stratum corneum of normal skin as well. Dectin 2 was detected in all layers of the epidermis in both fungal infections and in normal epidermis, too, without significant differences.
Discussion We performed a retrospective study on the expression of antimicrobial peptides and signalling molecules in lesional skin of human tinea and pityriasis versicolor. Some limitations need to be considered. A correlation with clinical data was not possible and presumably the biopsies studied came from lesions with different degrees of inflammation and had been taken at different phases of their development. Knowing this, no
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AMPs and TLRs in superficial mycoses
attempt was made to differentiate between early and acute lesions or between chronic and regressive ones. Furthermore, due to the fact that the biopsies were probably obtained during diverse clinical situations, a considerable heterogeneity of results was to be expected. Because a trustworthy grading of the intensity of immunostaining is not available, we decided to only discriminate between positive and negative staining. This is certainly not a very sensitive method, but in our opinion is adequate to the conditions, avoiding any pretence of an exactness that could not be ensured. Despite these restrictions, our findings clearly show that in superficial tinea as well as in pityriasis versicolor, an expression of defence-related antimicrobial peptides and of signalling molecules occurs. Our findings are in agreement with published data in infections with Candida and other fungi.9–14 The localization of the antimicrobial proteins within the upper epidermis also suggests a role in the defence against pathogens invading the skin from the outside. In view of the superficial localization of the agents, it is not surprising that the expression of these factors is mostly found in the upper epidermal layers. The antimicrobial peptides hBD2, hBD3 and psoriasin were scarcely detected in normal skin, but clearly upregulated in fungal infections. Interestingly, an occasional expression of hBD3 and psoriasin was confined to the stratum corneum only in normal skin, whereas in infected skin the staining extended to the stratum granulosum and stratum spinosum. Also, RNase7 was seen more often in the stratum granulosum of tinea compared with normal skin. The shift of the antimicrobial peptide expression to deeper layers of the epidermis was more pronounced in tinea compared with pityriasis versicolor, suggesting a correlation with the higher invasiveness of tinea. TLR2 and TLR4 can be expressed in normal skin, but are clearly more pronounced in fungal infection. In addition, a shift in the expression of TLR2 and TLR4 was noticed from the upper layers in normal skin to the lower epidermal layers in fungal infection. Again, the effects were more pronounced in tinea compared with pityriasis versicolor. This suggests that in severely infected skin, the innate immune defence already starts in the lower epidermis. We found no significant differences in the expression of TLR9 and dectin 2 in infected compared with normal skin. We conclude that the expression of TLR2 and TLR4 is very likely stimulated by the fungi, whereas a role of TLR9 and dectin 2 is uncertain.
© 2013 Blackwell Verlag GmbH
In view of the superficial localization of the pathogens, it is not surprising that the expression of the investigated factors is mostly found in the upper epidermal layers. The antimicrobial factors hBD2, hBD3 and psoriasin are clearly upregulated in the superficial fungal infections and they are mainly localised within the upper epidermis. It thus appears reasonable to hypothesise that these antimicrobial peptides and signalling molecules are involved in a stimulated defence reaction of the epidermis induced by the invaded fungi. Several factors of this system can have antifungal effects under certain conditions.5,15–18 We conclude from our findings that the innate immune system is activated in the process of superficial tinea and pityriasis versicolor by the causative fungi. Although in these infections, the fungi are mainly localised within the non-vital stratum corneum, it occurs that even in this remote ecological niche they are not out of reach for an antimicrobial defence mounted by living keratinocytes of the epidermis.
Conflicts of interest None
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