Acne is a disorder of the pilosebaceous unit resulting from multiple factors 1. Increased sebum production and follicular hyperkeratosis result in the development of microcomedones and in changes in the follicular milieu with intensive growth of Propionobacterium acne (Gram-positive anaerobic bacterium). P. acnes secrete several proinflammatory products that play an important role in the development of inflammation. These include lipases, proteases, hyaluronidases, and chemotactic factors. Immune responses to P. acnes include humoral and cell-mediated immunity, as well as complement activation 2.
The homologous family of transmembrane proteins termed toll-like receptors (TLRs) 3 is fundamental in the recognition of pathogen-associated molecular patterns (PAMPs) 4–6. The family of TLRs recognizes various PAMPs from different pathogenic origins such as bacteria, viruses, fungi, or protozoan parasites 4.
One of the TLR family members, the TLR4, is the key molecule of the lipopolysaccharide of Gram-negative bacteria-induced signaling 7, whereas the MyD88 (myeloid differentiation primary response protein 88) is the universal adaptor for TLRs and is also a member of the interleukin (IL)-1 receptor subfamily 8.
However, P. acnes were found capable of inducing TLR4 expression in vivo in the epidermis of acne lesions 9. Moreover, in humans, P. acnes have been shown to directly stimulate the production of proinflammatory cytokines by both human monocytic cell lines and freshly isolated peripheral blood mononuclear cells from acne patients and normal controls independent of phagocytosis or lipopolysaccharide contamination 10. The ability of P. acnes to bind both TLR2 and TLR4 on macrophages recruited at the site of inflammatory lesions will lead to the release of IL-12 and IL-18 locally 11–13.
As TLR4 activation was found to be essential for IL-18 secretion from macrophages in mouse models 14 and as we have previously shown elevated levels of IL-18 in inflammatory lesions of acne vulgaris 15, in this work we aimed to evaluate the level and role of TLR4 and its adaptor molecule MyD88 expression during the inflammatory and noninflammatory stages of acne development.
Patients and methods
This study included 60 patients with acne vulgaris and 30 age-matched and sex-matched apparently healthy controls recruited from the Dermatology Outpatient Clinic of Kasr Al-Eini Hospitals, Faculty of Medicine, and from the National Laser Institute of Cairo University. Inclusion criteria included age between 16 and 30 years and presence of at least five inflammatory and/or noninflammatory acne vulgaris lesions on the back. Exclusion criteria were: history of acne treatment within the past 3 months, patients on other treatments that are known to induce acne or comedogenic preparations, patients with other skin or systemic diseases, patients with occupational history of exposure to hydrocarbons, or pregnant women or lactating mothers.
Patients were classified into two groups: the inflammatory group including 30 patients with inflammatory lesions on the back and the noninflammatory group including 30 patients with comedonal lesions on the back.
A thorough history was obtained from all cases and a full dermatological examination was performed for all recruited individuals. A written informed consent was obtained from each of the patients and controls. Acne was graded as mild (<20 comedones, <15 inflammatory lesions, or total lesion count <30), moderate (20–100 comedones, 15–50 inflammatory lesions, or total lesion count of 30–125), or severe (>5 cysts, total comedone count >100, total inflammatory count >50, or total lesion count >125) 16.
A 2 mm punch skin biopsy of the following was obtained from the upper back of all individuals: inflammatory papules from the inflammatory group, comedones from the comedonal group, and normal skin from controls.
Total RNA was extracted from the skin biopsy using an RNA extraction kit (Qiagen, Valencia, California, USA). RNA quantity and quality were assessed by determination of the optical density at 260 and 280 nm using spectrophotometry.
The reverse transcription procedure was performed using SuperScript reverse transcriptase (Life Technologies Inc., Gaithersburg, Maryland, USA) and its accompanying reagents. Briefly, 2.5 mg of each tissue RNA was annealed to 0.4 mg of oligo-dT primer in a 12 μl volume. Measures of 4 μl of 5× buffer, 2 μl of 0.1 mol/l DTT, 1 μl of 10 mmol/l dNTP, and 1 μl of SuperScript reverse transcriptase were then added to bring the final reaction volume to 20 μl. After 1 h of incubation at 42°C, the reverse transcriptase mixture was incubated at 70°C for 10 min to inactivate the reverse transcriptase.
Two sets of primers were used for amplification of TLR4 and MyD88.
The TLR4 primers used for amplification reaction of the TLR4 were forward 5′-ATTCCTAAGGAAACCTGATTAAC-3′ and reverse 5′-GATATTAGCTTATAGGCAAGACG-3′ (NM_138554).
PCR reactions consisted of 25 pmol primers, 200 μmol/l dNTPs, 1.5 mmol/l MgCl2, 5 μl 10× PCR buffer, and 2.5 U Ampli Taq Gold DNA polymerase (Perkin-Elmer, Warrington, UK) in a total volume of 50 μl. A ‘hot start’ PCR was used, in which cycling conditions were as follows: 95°C enzyme activation for 12 min, followed by 1 min, 30 s at 94°C, 1 min at 56°C, and 1 min at 72°C for 35 cycles and a final extension of 10 min at 72°C.
The MyD88 primers used in the amplification process were forward 5′-CAGGATGCAAGATATATTCCAGG-3′ and reverse 5′-ATTTTAAAGCCATCTCAAGAGGC-3′ (NM_002046).
The PCR amplification was carried out for 30 cycles at 95°C for 30 s, at 54°C for 30 s, and at 72°C for 30 s. All the PCR products were electrophoresed on a 2% agarose gel stained with ethidium bromide and visualized by an ultraviolet transilluminator. Gene expression of TLR4 produced sharp bands at 125 bp and that of MyD88 produced bands at 148 bp (Figs 1 and 2, respectively).
The PCR products were then quantitated using a quantitation kit (Promega Corporation, Madison, Wisconsin, USA). This method depends on purification of the PCR using Promega Wizard PCR preps DNA purification kit (Promega Corporation). The mixture for quantitation consisted of DNA quantitation buffer, sodium pyrophosphate, nucleoside diphosphate kinase enzyme solution, T4 DNA polymerase, and DNA. All these contents were incubated at 37°C for 10 min. Thereafter, 100 μl of Enliten L/L reagent (Promega Corporation, Madison, USA) was added. Immediately, the reaction was read using a luminometer. The same steps were carried out on DNAs of known concentrations provided by the kit, and a standard curve was plotted using the readings of the luminometer against the concentrations. Then, the readings of the amplified PCR product of TLR4 and My88 after using the luminometer were read from the standard curve. The results were expressed as µg/g tissue (Figs 1 and 2).
Data were statistically described in terms of range, mean SD, median, frequencies (number of cases), and percentages when appropriate. Comparison of quantitative variables between the study groups was made using the Student t-test for independent samples for comparing two groups when normally distributed and the Mann–Whitney U-test for independent samples when not normally distributed. Comparison of quantitative variables between more than two groups in the present study was made using the one-way analysis of variance test with least significant difference post-hoc multiple two-group comparisons when comparing normally distributed data. Comparison of quantitative variables between more than two groups of non-normal data was made using the Kruskal–Wallis analysis of variance test with the Mann–Whitney U-test for independent samples as post-hoc multiple two-group comparisons. For comparing categorical data, the χ2-test was used. The exact test was used when the expected frequency was less than 5. Correlation analysis between variables was carried out using the Pearson moment correlation equation for linear relation. All statistical calculations were made using computer programs Microsoft Excel 2003 (Microsoft Corporation, New York, New York, USA) and Statistical Package for the Social Science (SPSS; SPSS Inc., Chicago, Illinois, USA) version 15 for Microsoft Windows.
The total number of female participants in the study was 42 (46.7%). The number of female participants was 29 in the patient group (12 in the inflammatory and 17 in the comedonal group) and 13 in the control group. The total number of male participants in the study was 48 (53.3%). The number of male participants was 31 in the patient group (18 in the inflammatory and 13 in the comedonal group) and 17 in the control group. The ages of all recruits in this study ranged from 16 to 27 years with a mean of 20.21±3.182 years. The mean age in the patient group was 19.87±3.039 years (19.93±2.66 for the inflammatory and 19.8±3.41 for the comedonal group) and was 20.90±3.397 years in the control group. There was no significant difference between the two groups (patients and controls) with regard to age and sex (P=0.823 and 0.165, respectively). Also, age and sex distributions between the inflammatory and comedonal subgroups showed no significant difference (P>0.05).
Acne severity in patients showed 29 mild cases (48.3%; 12 in the inflammatory and 17 in the comedonal group), 26 moderate cases (43.3%; 16 in the inflammatory and 10 in the comedonal group), and five severe cases (8.3%; two in the inflammatory and three in the comedonal group). According to patient history there were 16 stationary cases (26.7%), 14 progressive cases (23.3%), and 30 cyclic cases (50%). In the inflammatory group, there were eight stationary, eight progressive, and 14 cyclic cases, whereas in the comedonal group there were eight stationary, six progressive, and 16 cyclic cases.
TLR4 mRNA levels were statistically significantly higher in patients compared with controls (P<0.001). The TLR4 mRNA values for patients ranged from 751 to 1346 μg/g of tissue with a mean value of 986.67±131.904 μg/g tissue, whereas for controls the TLR4 mRNA values ranged from 426 to 898 μg/g of tissue with a mean value of 755.87±123.609 μg/g tissue (Fig. 3). The mean value of TLR4 mRNA was higher in inflammatory lesions (1071.73±127.111 μg/g tissue) than in comedones (901.60±65.331 μg/g tissue), and this was statistically significant (P<0.001).
The mean values of TLR4 mRNA of all recruited patients were highest in patients with progressive course in comparison with those with cyclic and stationary course of disease (mean±SD: 1083.143±113.118; 970.567±137.479, and 932.438±91.988, respectively). However, this elevation was only statistically significant between progressive and stationary courses (P<0.001) (Fig. 4).
MyD88 mRNA levels were higher in patients in comparison with controls in a statistically significant manner (P<0.001). The MyD88 mRNA values for patients ranged from 524 to 1013 μg/g tissue with a mean value of 866.58±118.223 μg/g tissue, whereas for controls the MyD88 mRNA values ranged from 402 to 846 μg/g tissue with a mean value of 605.90±130.558 μg/g tissue (Fig. 3). The mean value of MyD88 mRNA was higher in inflammatory lesions (922.00±68.189 μg/g tissue) than in comedones (811.17±132.029 μg/g tissue), and this was statistically significant (P<0.001).
The mean values of MyD88 mRNA of all recruited patients were highest in patients with progressive course than in those with cyclic and stationary courses (mean±SD: 928.357±76.4; 887.967±88.757, and 772.438±144.1, respectively). However, none of these comparisons showed any statistical significance (Table 1).
Correlations between TLR4 and MyD88 and clinical data
There was a weak correlation between TLR4 and MyD88 mRNA in only the comedonal group, and this resulted in an overall weak statistically significant correlation between TLR4 and MyD88 mRNA in all (inflammatory+comedonal) patients (Pearson’s correlation=0.456, P<0.01) (Fig. 5). There was no statistically significant correlation between TLR4 and MyD88 mRNA in controls (Pearson’s correlation=−0.310, P=0.096). There was no significant correlation between TLR4 or MyD88 mRNA and disease duration in either group, in which Pearson’s correlations were −0.178 (P=0.175) for TLR4 and 0.226 (P=0.083) for MyD88.
The results of the current study demonstrated a significant upregulation of TLR4 and its adaptor protein MyD88 in acne vulgaris lesions in comparison with controls and also showed that this upregulation is affected by the stage of development of the acne lesions.
In acne, the increased sebum production and follicular hyperkeratosis result in the development of microcomedones and in changes in follicular milieu with intensive growth of P. acnes. P. acnes secrete several proinflammatory products, which play an important role in the development of inflammation. These include lipases, proteases, hyaluronidases, and chemotactic factors. Immune responses to P. acnes include humoral and cell-mediated immunity, as well as complement activation 2. Moreover, P. acnes are capable of inducing TLR2 and TLR4 expression in vivo in the epidermis of acne lesions 9.
Several adaptor proteins for TLR have been described. MyD88 is the universal adaptor for TLRs that signal through the TIR domain 8, whereas MyD88 adaptor-like protein (Mal) acts as a bridging adaptor for MyD88 but only in the context of TLR2 and TLR4 stimulation 17. MyD88, TLR2, and TLR4 are largely electropositive on their surfaces; hence, MyD88 is unable to bind these TLRs. Mal, in contrast, is mainly electronegative on its surface, allowing it to bind TLR2 and TLR4 and bring MyD88 into the signaling complex 18. It is now known that two signaling pathways exist following TLR4 activation: the MyD88-dependent and MyD88-independent pathways. These signaling pathways depend on TIR adaptor proteins including MyD88, Mal/TIRAP (MyD88 adaptor-like protein/toll/interleukin-1 domain-containing adaptor protein), TRIF/TICAM-1 (TIR domain-containing adaptor protein inducing IFNβ/TIR-containing adaptor molecule-1), and TRAM/TICAM-2 (TRIF-related adaptor molecule/TIR domain-containing adaptor molecule 2) 19. Once activated by a PAMP, a TLR triggers a cascade of cellular signals, culminating in the eventual activation of NF-κB, which binds to a discrete nucleotide sequence in the upstream regions of genes that produce proinflammatory cytokines such as TNFα, IL-1, and IL-2, thereby regulating their expression. The release of these cytokines such as IFNγ is the hallmark of the cellular response to the activation of the innate immune system 20.
The work of Kim et al.21 and Jarrousse et al.22 showed that P. acnes may induce the expression and activation of TLR2 in acne vulgaris. In our study, using reverse transcriptase-PCR the levels of TLR4 mRNA were significantly higher in inflammatory and noninflammatory acne lesions compared with controls (P<0.001). The level of MyD88 mRNA was also significantly higher in patients in comparison with controls (P<0.001). Supporting these findings, Jugeau et al.9, who studied the effect of P. acnes upon TLR activation in keratinocytes, reported through immunohistochemical analysis that, in vivo, TLR2 and TLR4 expression levels were increased in the epidermis of acne lesions. However, they only examined epidermal TLR4 expression in inflammatory acne lesions; TLR4 is now known to be expressed on human sebaceous glands 23,24 and on murine hair follicle epithelia 25. Moreover, Nagy et al.26 found that two of four clinical isolates of P. acnes significantly induced human β-defensin-2 mRNA expression and that all four strains significantly induced IL-8 expression; all of these effects could be inhibited by anti-TLR2-neutralizing, and anti-TLR4-neutralizing antibodies.
This increased expression of TLR4 mRNA that we detected in both inflammatory and comedonal acne lesions is most likely due to P. acnes colonization, which according to Jugeau et al.9 may occur during the first hour of incubation with P. acnes.
Although we found an elevation of MyD88 level in acne lesions, our results showed that there is a weak correlation between this protein and the level of TLR4. This implies that TLR4 signaling in acne vulgaris lesions might be mediated through different mechanisms, possibly through MyD88-independent pathways. TRIF, which acts as the adaptor for the MyD88-independent pathway induced by TLR4 27, seems to be a potential candidate for activating the TLR4 response in acne vulgaris.
We observed that both TLR4 and MyD88 mRNA expression levels were significantly higher in inflammatory lesions than in comedonal lesions and were higher in patients with a progressive course than in those with a cyclic or stationary course. Our findings suggest that TLR4 and MyD88 are upregulated with the evolution of lesions from comedone to inflammatory papules and that TLR4 is upregulated with progression in the course of the disease. Interestingly, when we examined the inflammatory lesions alone, their MyD88 expression was not upregulated by progression of the course of acne. MyD88 expression does not seem to be affected by the course of disease in inflammatory papules. Again, this questions its role in mediating TLR4 signaling in acne vulgaris. However, there are two limitations regarding the course of the disease and its relation to the expression of both proteins. First, the course parameter in acne vulgaris measures the course of the whole disease and not merely the individual lesion, which was used to examine for TLR4 and MyD88 expression. Second, it is difficult to determine whether the cyclic patients in both groups were experiencing an exacerbation or a remission at the time the sample was obtained.
Our findings, together with previous reports, implicate P. acnes in the upregulation of TLR4 in early comedonal acne vulgaris lesions, and this upregulation is exaggerated as the lesion progresses into an inflammatory papule. We propose that TLR4 may be directly involved in the pathogenesis and progression of acne vulgaris lesions. On the basis of the results obtained, we suggest that MyD88 is also upregulated in acne lesions, corresponding, even though weakly, to the TLR4 amplification. The degree of involvement of MyD88-independent pathways in mediating the TLR4 signaling in acne vulgaris may be worth further investigations.
The diversity of the skin’s antimicrobial peptide and cytokine production in reaction to P. acnes colonization may influence the clinical course of acne, and identifying this immune response may help in a better understanding of acne and in the development of more specific therapies.
Conflicts of interest
There are no conflicts of interest.
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