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7DIVERSE KERATINOCYTE EXPRESSION OF TLR2, HBD-1 AND HBD-2 IN ROSACEA
O.A. Svitich
professor, MD, PhD, Mechnikov research Institute of vaccines and sera, Moscow
L.V. Gankovskaya professor, MD, PhD
Pirogov Russian National Research Medical University, Moscow
I.V. Khamaganova professor, MD, PhD
Pirogov Russian National Research Medical University, Moscow
O.L. Novozhilova
doctor, Moscow scientific & practical centre for dermatovenereology & cosmetology
ABSTRACT
the objective of the study was the complex analysis of indicators of innate immunity of the keratinocytes in healthy persons and patients with rosacea. The participants: 35patients with rosacea aged 21- 72 years old, including 8 men, 27 women. The entry criteria were erythematotelangiectatic & papulopustular subtype of rosacea. The exclusion criteria were pregnancy, breast-feeding and proved malignancies. Results: The statistically significant difference was revealed in the gene expression in rosacea patients in comparison with healthy subjects. The expression of Toll-like receptor 2(TLR2) gene in keratinocytes patients with rosacea without Demodex both in healthy and pathological areas observed a significant decrease in gene expression of TLR2. In patients with rosacea and Demodex expression of TLR2 did not drop as critical. Expression of hBD-1 in keratinocytes of patients with rosacea without Demodex expression level of defensin decreased. In patients with Demodex and rosacea level of gene expression of Human beta defensin-1 (hBD-1) was reduced significantly. The indicator gene expression of Human beta defensin-2(hBD- was reduced significantly in different groups as well. Conclusion: the innate immunity in rosacea patients differs significantly from the innate immunity in healthy subjects.
Keywords: rosacea, innate immunity of the keratinocytes, antimicrobial peptides, beta- defensins.
Анализ многолетнего опыта функционирования программ npInroduction
Rosacea is a common inflammatory facial skin disease, characterized by erythema, telangiectasia, papules and pustules. Rosacea often begins with flushing and progresses to persistent redness and the appearance of visible blood vessels. [1,2].The 4 major clinical subtypes of rosacea are generally applicable: erythematotelangiectatic rosacea, papulopustular rosacea, phymatous rosacea, and ocular rosacea. Steinhoff M et al.[3] emphasized that it needed to be elucidated whether these subtypes develop in a consecutive serial fashion or if any subtypes might occur individually as part of a syndrome. Because rosacea often affects multiple family members, a genetic component is also suspected, but the genetic basis of rosacea remains unclear. During disease manifestation and early stage, the innate immune system and neurovascular dysregulation seem to be driving forces in rosacea pathophysiology. Dissection of major players for disease progression and in advanced stages is severely hampered by the complex activation of the innate and adaptive immune systems, enhanced neuroimmune communication, profound blood vessel and possibly lymphatic vessel changes, and activation of almost every resident cell in the skin. [3] . Many mechanisms interact with the cutaneous innate immune system that may be operative. A variety of potential triggers stimulate this immune detection system which is upregulated and hyper-responsive in facial skin of patients with rosacea as compared to normal skin. The presence of a microbial organism is not a primary or mandatory component of the pathogenesis of rosacea. [4] Skin sample analysis showed a higher expression of genes encoding pro-inflammatory cytokines (Il-8, Il- 1b, TNF-a) and inflammasome-related genes (NALP-3 and CASP-1) in rosacea, especially PPR. Overexpression of LL-37 and VEGF, as
well as CD45RO, MPO and CD163, was observed, indicating broad immune system activation in patients with rosacea. [5] Recent molecular studies suggest that an altered innate immune response is involved in the pathogenesis of the vascular and inflammatory disease seen in patients with rosacea. These findings may help explain the benefits of current treatments and suggest new therapeutic strategies helpful for alleviating this disease.[6] Several molecular features of its inflammatory process have been identified: an overproduction of Toll-Like receptors 2, of a serine protease, and of abnormal forms of cathelicidin. The two factors which stimulate the Toll-like receptors to induce cathelicidin expression are skin infection and cutaneous barrier disruption: these two conditions are, at least theoretically, fulfilled by Demodex, which is present in high density in PPR and creates epithelial breaches by eating cells. [1]Keratinocytes are an immunological barrier against infection. Together with lymphoid tissue they participate in the innate immune system by performing the following functions: 1) removal of large particles and microorganisms, 2) recognition pathogens through pattern recognition receptors -TLRs, 3), the secretion of inflammatory mediators cytokines and antimicrobial peptides.[7].hBD-1 is constitutively expressed in keratinocytes, but exhibits only minor antibiotic killing activity in comparison with other defensins[8,9]. The reduced form of hBD-1 was shown to become a potent antimicrobial peptide, of which reduction was catalyzed by thioredoxin expressed in the epidermis [10] .This suggests that the redox regulation is crucial for the innate immune protection by hBD-1. Elevated hBD-2 expression in human keratinocytes exposed to pathogenic bacteria was described[7]Gallo RL and Nakatsuji T highlighted that our resident commensal microbes produced their own AMPs, act to enhance the normal production of AMPs by keratinocytes, and were beneficial to maintaining inflammatory
homeostasis by suppressing excess cytokine release after minor epidermal injury. [11] These observations indicate that the normal human skin microflora protects skin by various modes of action, a conclusion supported by many lines of evidence associating diseases such as acne, atopic dermatitis, psoriasis, and rosacea with an imbalance of the microflora even in the absence of classical infection.
The objective of the study was the complex analysis of indicators of innate immunity of the keratinocytes in healthy persons and patients with rosacea. We evaluated TLR2, hBD-
1 and hBD-2 expression at the mRNA levels in keratinocytes from patients with rosacea.
Material & methods 35 patients with rosacea aged 21- 72 years old, including 8men, 27 women were examined in the Moscow Research and Practical Center for Dermatovenereology and Cosmetology.
2 patients had erythematotelangiectatic subtype of rosacea, in 1 patient D. folliculorum was detected, in 1 - was not detected.15patients had papulopustular subtype of rosacea, in 9 patients D. folliculorum was detected, in 6 - was not detected.
6 healthy subjects aged 30- 39 years old were examined.
Samples of keratinocytes from patients were collected using swabs that were placed into vials containing 1 ml of polymerase chain reaction (PCR) transport medium and kept at - 200C until being processed for gene expression by PCR (see below).
Isolation of RNA and RT- PCR Analysis
Total RNA was isolated from keratinocytes by using anRNeasy Mini Kit (Qiagen, Germany) and kit "RIBO-sorb" (ILS, Russia) according to the manufacturer's instructions. For cDNA synthesis, 2 ^l of total RNA was combined with random primers (Syntol, Russia) and reverse primers for the target genes (T2rev, B1rev, B2rev). The mix was heated to 75oC for 3 min and then quick-chilled on ice; 3 ^l of 10X Reverse Transcriptase Buffer (SibEnzime, Russia), 2 ^l of 2.5mM dNTP (SibEnzime, Russia) and 10 ^l of M-Multi Reverse Transcriptase (SibEnzime, Russia) were added to the mix (final volume of 30 ^l). Afterwards, the mix was kept at 37oC for 60 min and at
95oC for 10 min. [12,13]
For quantitative analysis, real-time PCR was performed, using a SYBR Green Kit for qRT-PCR (Syntol, Russia), according to the manufacturer's instructions. The primers and probes for the real-time PCR were synthesized by Syntol (Russia). The PCR reactions were carried out at 50°C for 2 min, 95°C for 2 min, followed by 40 cycles at the primer-specific annealing temperature (60°C) for 50s and 95°C for 20s. The mRNA expression levels of the target genes were normalized with the expression of the housekeeping gene ^-actin[14] Statistical analysis
Statistical differences of the gene expression levels were determined by using a Mann-Whitney U test. p < 0.05 was considered to be statistically significant. The results are shown as the mean ± SEM. Results
We applied an analytical approach in order to evaluate the innate immunity of the skin. It included the definition of gene expression of the receptors - TLR2 and antimicrobial peptides hBD1 and hBD2. Thus, we have shown that the keratinocytes of the skin of donors expresses genes of the innate immunity TLR2, hBD1 and hBD2.
The statistically significant difference was revealed in the gene expression in rosacea patients in comparison with healthy subjects.
The expression of TLR2 gene in keratinocytes in the group of healthy subjects was 16489+/-3553 copies of the TLR2 gene (relative to the expression of actin). In the group of patients with rosacea without Demodex both in healthy and affected areas observed a significant decrease in gene expression of TLR2.(Fig.1)
Expression of hBD-1 in keratinocytes of healthy subjects was 92943+/-27645 number of copies of the test gene. In patients with Demodex and rosacea level of gene expression of hBD-1 was reduced more critical than in patients without Demodex.(Fig.2)
The level of gene expression of hBD-2 reduced both in patients with Demodex and rosacea and in patients without Demodex and rosacea.(Fig3)
Fig.1. The expression of TLR2 gene in healthy subjects, healthy and pathologic areas in patients with rosacea & demodicosis
Fig.2. The expression of hBD-1 gene in healthy subjects, healthy and pathologic areas in patients with rosacea &demodicosis
Fig.3. The expression of hBD-2 gene in healthy subjects, healthy and pathologic areas in patients with rosacea & demodicosis
Discussion
Nowdays we have substantial evidence of the dysregulation of immune system in rosacea.[1,3- 6].The altered innate immune response in rosacea patients is proved by the presented complex analysis of indicators of innate immunity of the keratinocytes in healthy persons and patients with rosacea. Our investigation showed statistically significant decrease of the indicators of innate immunity(TLR2, hBD-1, hBD-2) in rosacea patients. The obtained results suggest immunodeficient state in rosacea patients.
In conclusion, now we have an obvious question of therapy of rosacea. An appropriate immunotropic treatment is to be worked out .Moreover, we must evaluate the immune effects in cases of successful treatment of rosacea.
We declare no conflict of interests.
There was no funding of our work.
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