Introduction

Acne vulgaris is a common disease that caused skin inflammation in pilosebaceous skin tissue. It occurs about 80–90% the people within 11–30 years old and 5% of people older than 30 years old [1]. The pathogenesis of acne vulgaris is multifactorial as a result of the proliferation of Propiobacterium acnes and inflammation [2]. The population of P. acnes on the skin activates the immune response through Toll-like receptor (TLR)-2 induction to polymorphonuclear monocyte cell (PMN) around the sebaceous follicle to secrete proinflammatory cytokines such as interleukin (IL)-1α, IL-8, and tumor necrosis factor (TNF)-α [3, 4]. A recent study revealed that P. acnes could release various proteases through the activation of protease-activated receptor (PAR)-2 inside the keratinocyte that increases the transcription of the proinflammatory cytokine, including IL-1α, IL-8, and TNF-α. Moreover, the metalloproteinase matrix and LL-37 which known as a group of anti-microbes cathelicidin peptides [5].

TNF-α and IL-1α induce the secretion of IL-8, an inflammatory mediator capable of increasing the paracrine inflammatory signal, through the recruitment of neutrophil to the active lesion. Enzymes released by neutrophils lead to the bursting of epithelium follicles and inflammation [2, 6, 7]. The IL-1α involved in the acne pathogenesis through the inflammatory reaction and skin tissue damage. The IL-8 mediates paracrine inflammation via the chemokine CXC, which increases the inflammatory signal through demargination, activation, and chemotaxis from polymorphonuclear leukocytes [8]. The pleiotropic cytokine that positively functioned affects the various kinds of cell and it is considered essential for connection between immune response and adaptive [9].

Correlation among levels of TNF-α, IL1-α, and IL-8 in acne vulgaris severity has been addressed as the subject of several studies. Szabo et al. reported a correlation between a polymorphism of the gene that encodes TNF-α and acne vulgaris severity. Genes with higher polymorphism of minor allele tend to increase the severe inflammation of acne. On the other hand, gene polymorphisms are not related to the acne vulgaris [9]. Another study by Hussain et al. showed that there is a significant increase in the plasma level of IL-8 in acne vulgaris patients compared to the normal subjects (p<0.0001) [10].

This study aims to determine the correlation between the plasma levels (TNF-α, IL1-α, and IL-8) and acne vulgaris severity. Our study suggested that there is no evidence supporting the correlation of TNF-α, IL1-α, and IL-8 plasma levels with the acne vulgaris severity, but it only demonstrated a general correlation with acne vulgaris.

Materials and Methods

The research was conducted by using observational cross-section in the Saiful Anwar General Hospital, Malang, Indonesia. Seventy-eight patients of were Dermatology and Venereology were addressed as a subject for the research based on certain criteria. Samples were collected by consecutive sampling during January—April 2016.

There were an inclusion and exclusion criteria for the patient. The inclusion criteria for acne vulgaris patients were: physically healthy, aged 15–30 years old, and provided signed informed consent. Then, the exclusion criteria were related to condition of patient, such as pregnant, breastfeeding, menstruating, and hormonal contraception used in women [11]. Additional exclusion criteria were including patients who had injected or consumed several substances within two weeks before the study. The substances were topical antibiotics, benzoyl peroxide, tretinoin, adapalene, nicotinamide, salicylic acid, and tea-tree oil [12]. However, treatment with the following medicines can affect the inflammation of acne vulgaris: oral antibiotics, oral retinoids, antiandrogens, and consuming anti-inflammation for a month [13]. The exclusion criteria also included smoking, systemic disease, other skin diseases, and patients with a fat mass of more than 30%.

Three members of the research team examined all patients on the same day. The acne severity was determined based on the combine acne severity classification (CASC) which divided acne severity into a mild, medium, and severe [14]. The vena blood samples obtained from subjects and plasma were stored at –20 °C before further analysis. The plasma levels of TNF-α, IL-1α, and IL-8 were analyzed by enzyme-link immunosorbent assay (ELISA) in the Biomedics Centre Laboratory, Medical Faculty, Brawijaya University.

Data normality was analyzed based on the Kolmogorov—Smirnov test. The correlation among the plasma levels of TNF-α, IL-1α, and IL-8 with acne severity was examined by Spearman correlation test. This research was accepted by the ethics committee of Dr. Saiful Anwar General Hospital (400/17/K.3/302/2015), as mentioned in the Helsinki Declaration.

Results

Demographic characteristics

Seventy-eight patients with acne vulgaris consist of 37 men (47.43%) and 41 women (52.54%); all patients are 13–35 years old. The result showed that there is no significant difference between acne vulgaris and age (p<0.05). A similar result also demonstrated that acne vulgaris and gender has no significant difference (table 1).

Таблица 1. Характеристика пациентов по возрасту, полу и степени тяжести акне

Characteristic	Category	Acne vulgaris severity						p-value
		mild		moderate		severe
		freq	%	freq	%	freq	%
Age (years)	13–19	0	0	4	5.1	11	14.1	0.00
	20–34	26	33.3	22	28.2	15	19.2
Gender	Men	9	11.5	16	20.5	12	15.4	0.149
	Women	17	21.8	10	12.8	14	17.9

The Spearman correlation coefficient values of IL-8, TNF-α, and IL-1α serum levels and acne vulgaris are 0.007 (p=0.952; p>0.05), –0.088 (p=0.442; p>0.05), and 0.158 (p=0.158; p>0.05), respectively. Based on these values, there is no significant correlation between the plasma levels of IL-8, TNF-α, and IL-1α and acne vulgaris severity (table 2).

Таблица 2. Значения корреляции Спирмена между уровнями ФНО-α, ИЛ-1α и ИЛ-8 в плазме крови и тяжестью акне

Plasma level	Correlation coefficient (r) of acne vulgaris severity	p-value
TNF-α	–0.088	0.442
IL-1α	0.158	0.168
IL-8	0.007	0.952

There was no significant correlation between the plasma levels of TNF-α, IL-1α, and IL-8 and acne vulgaris severity (fig. 1, a–c).

Рис. 1. Статистическая линейная зависимость между тяжестью акне и уровнями (а) ИЛ-8, (б) ФНО-α и (в) ИЛ-1α.

Correlation between TNF-α and IL-1α

The correlation coefficient between TNF-α and IL-1α was 0.078 (p=0495; p>0.05). There was no significant correlation between IL-1α and TNF-α (table 3).

Таблица 3. Значение корреляции Спирмена между уровнями ФНО-α и ИЛ-1α

Cytokine	Correlation coefficient of IL-1α level	p-value
TNF-α level	0.078	0.495

The line in fig. 2 represents the correlation between the level of TNF-α and IL-1α which does not show a clear trend. It is indicating that there is no significant correlation.

Рис. 2. Линейная зависимость между уровнями ФНО-α и ИЛ-1α.

Correlation between IL-1α and IL-8

The correlation coefficient between IL-1α and IL-8 is 0.187 (p=0.102; p>0.05; table 4). There is no significant correlation between IL-1α and IL-8.

Таблица 4. Значение корреляции Спирмена между уровнями ИЛ-1α и ИЛ-8 (логарифмическая)

Cytokine	Correlation coefficient of IL-8	p-value
IL-1α level	0.187	0.102

The line in fig. 3 does not show a clear trend. Therefore, there is no significant difference between IL-1α and IL-8.

Рис. 3. Линейная зависимость между уровнями ИЛ-8 (логарифмическая) и ИЛ-1α.

Correlation between TNF-α and IL-8

The correlation coefficient between TNF-α and IL-8 is 0.193 (p=0.090; p>0.05; table 5). It shows that there is no significant correlation between TNF-α and IL-8.

Таблица 5. Значение корреляции Спирмена между уровнями ФНО-α и ИЛ-8

Cytokine	Correlation coefficient of IL-8	p-value
TNF-α level	0.193	0.090

The line between TNF-α and IL-8 is nearly horizontal indicating that there is no significant correlation between TNF-α and IL-8 (fig. 4).

Рис. 4. Линейная зависимость между уровнями ИЛ-8 и ФНО-α.

Discussion

The age range of patients in this study is 15–30 years old, while acne vulgaris usually affects individuals from 11–30 years old [1, 15]. The highest prevalence of patients with acne vulgaris in the United States is on average 25 years old. Teenagers (13–19 years old) have higher risks of severe acne vulgaris compared with adults [15].

Acne vulgaris lesions tend to be severe in teenagers but it recovers over time. The number of acne vulgaris cases will increase in the mid-20s. However, in some cases, it can be found at 40-year-old patients as a result of hormonal problems [15, 16]. According to the World Health Organization (WHO), the hormonal changes in teenager between 12 and 24 years old are related to puberty. It happened when the gonads produce increased levels of androgen hormones [17].

The increased level of androgen hormones in teenagers induce the stimulation of the pilosebaceous glands. Acne vulgaris can also be caused by non-hormonal factors. Several types of acne are not affected by dehydroepiandrosterone sulfate. Therefore, acne can occur in adults after puberty which indicated by Propionibacterium acnes in the adult skin with an increased number of sebaceous glands. The population of P. acnes increases dramatically during puberty and stable in adults.

In this study, acne vulgaris is more common in women than in men (52.6%). However, there is no significant difference in the severity of acne vulgaris between man and women. A previous study reported that the number of cases of acne vulgaris is higher in women than men [18]. The sex factor in acne severity has been reported in several studies with various results. Hormonal factors are expected to be the dominant acne precursor in women because hormones regulate sebum secretion.

The involvement of TNF-α, IL-1α, and IL-8 in the pathogenesis of acne vulgaris is primarily in lesion inflammation due to P. acnes binds TLRs in macrophages causing the activation of NF-kB and secretion of TNF-α, IL-1α, and IL-8 followed by formation of the inflammatory lesions of acne vulgaris [2, 9]. The limitation of this study is the Lehmann method of acne severity examination, which calculates the total number of lesions compared with inflammatory and non-inflammatory lesions [14]. This leads to difficulty in examining lesions due to acne vulgaris inflammation only. Another acne severity scoring method was introduced by Plewig and Kligman which calculates the number of blackheads and inflammatory lesions separately based on total and lesion types [16]. The determination of acne severity by separating inflammatory and non-inflammatory lesions is accurate in determining acne vulgaris severity if inflammation is the focus of the study.

TNF-α, IL-1α, and IL-8 are pro-inflammatory cytokines that are systemically affected by several factors; i.e., infection. They function as paracrine effectors by affecting the surrounding cells and local mediators, so that the presence of these pro-inflammatory cytokines can exert different effects systemically compared with locally [11]. It can explain why the examination of local cytokine production at locations of inflammation is biologically more relevant than the study of cytokine production in the peripheral bloodstream.

IL-8 increased with increases in body mass index. Increased adipose tissue in obese patients results in higher risks due to increased production of various pro-inflammatory cytokines, including TNF-α, IL-1α, and IL-8. Obesity reflects a chronic inflammatory state [19]. Plasma levels of TNF-α, IL-1α, and IL-8 in the pre-obesity and obesity patient are significantly higher than non-obese patients (p<0.001). It is related to the high-fat percentage (fat mass >30%) as an exclusion criterion this study [20].

Other conditions related to high levels of TNF-α, IL-1α, and IL-8 are diseases based on T-cell helper 2 domination including asthma and atopy. A case-control study reported that Th1 cells and its cytokine in asthma cases, through Th-17 derivate lymphocyte, will increase in the inflamed air tube and produced IL-17. Pro-inflammatory cytokine IL-17 induced other pro-inflammatory cytokines, such as TNF-α and IL-8 which are related to asthma severity. Silvestri et al. reported that serum levels of IL-8 and TNF-α were higher in severe asthma patients compared with healthy controls [21].

The results of this study support previous studies that also reported no significant correlation between plasma levels of TNF-α, IL-1α, and IL-8 and acne severity. Previous research by Ingham et al. described the correlation between TNF-α and IL-1α towards Propionibacteria sp., Staphylococci sp., and Malassezia sp. in the opened blackheads. This study reported that there is no significant correlation. Moreover, this study also reported that IL-1α was found in the opened comedo and that it was responsible for the disruption of sebaceous follicle walls and inflammation [22].

Acknowledgement

Authors thank the Brawijaya University, Malang, Indonesia, for the funding of this research.

Author’s contribution

Authors: S. Prawitasari, T. Sugiman, A. Widiatmoko, D.P. Ekasari

The concept and design of the study: S. Prawitasari, T. Sugiman

Collecting and interpreting the data: S. Prawitasari, A. Widiatmoko

Statistical analysis: A. Widiatmoko, D.P. Ekasari

Drafting the manuscript: S. Prawitasari, T. Sugiman

Revising the manuscript: A. Widiatmoko, D.P. Ekasari