Evaluation of Serum Levels of Interleukins 6, 8, 17 and 22 in Acne Vulgaris: A Cross-Sectional Study

Abstract

Background:

Acne vulgaris (AV) is a chronic, multifactorial, inflammatory skin disease, and it is now becoming increasingly clear that the inflammatory pathway is involved at a very early in the pathogenesis of acne. The Th17 cells, the activators of this cell line and its downstream effector cytokines, are all likely to have a critical role in inducing and maintaining the disease.

Aim:

To analyse the role of interleukins (ILs) 6, 8, 17 and 22 in the pathogenesis of acne.

Materials and Methods:

Sixty patients of AV and thirty age- and sex-matched controls were included in our study. Serum levels of interleukins 6, 8, 17 and 22 were determined using an enzyme-linked immunosorbent assay (ELISA), and thereafter, levels were correlated with the severity of acne.

Result:

Serum levels of IL-6, IL-8, IL-17 and IL-22 were 0.15 ± 0.0174 pg/ml, 0.38 ± 0.080 pg/ml, 0.19 ± 0.0075 pg/ml and 0.23 ± 0.0152 pg/ml in cases, respectively, and 0.13 ± 0.0095 pg/ml, 0.14 ± 0.034 pg/ml, 0.13 ± 0.0033 pg/ml and 0.21 ± 0.0099 pg/ml in controls, respectively. The difference in levels between cases and controls was significant for IL-8 and IL-17, while for IL-6 and IL-22 the difference was insignificant. There was a highly significant positive correlation between IL-8 and IL-17 levels. IL-6 and IL-8 showed a significant positive correlation with the severity of disease.

Conclusion:

IL-8 and IL-17 play a critical effector role in the pathogenesis of AV. IL-6-stimulated Th17 cells are likely the major producers of IL-8 in acne lesions.

Introduction

Acne vulgaris (AV) is a chronic, multifactorial, pleomorphic inflammatory skin disease.[1] The pathogenesis of acne involves the following processes: increased sebum production, Cutibacterium acnes (C. acnes)—formerly known as Propionibacterium acnes colonisation, hyper-cornification and inflammation of the pilosebaceous duct.[2] The exact sequence of the above-mentioned events and how are they interconnected is not clearly understood. It has been suggested that sebocytes in acne have an exaggerated response to androgens, which leads to hyperplasia of sebaceous glands along with increased sebum production.[3] C. acnes colonises the follicular duct and breaks down sebum triglycerides to free fatty acids.[4] Free fatty acids stimulate NF-κB nuclear translocation in keratinocytes to release IL-6, IL-1β and tumour necrosis factor α (TNF-α).[3] IL-6 functions as a pro-inflammatory cytokine and induces hyperkeratosis of pilosebaceous duct.[3] In addition, C. acnes activates the innate immune system through Toll-like receptors (TLRs) 2 and 4 present on keratinocytes and monocytes to release IL-1, IL-6, IL-8, IL-12, TNF-α and matrix metalloproteinases.[2]

Along with innate immunity, adaptive immunities such as Th1 and Th17 pathways also play a role in pathomechanism in acne. Naïve CD4+ cells differentiate into T helper (Th1 and Th17) cells based upon local ’cytokine milieu’ before their functional activities and release IFN-ϒ, IL-17 and IL-22.[5]

We hypothesised that serum levels of interleukins 6, 8, 17 and 22 are altered in AV patients. In this study, serum level of IL-6, IL-8, IL-17 and IL-22 cytokines is measured and their correlation with the severity of acne has been assessed.

Materials and Methods

A cross-sectional study was conducted in the Department of Dermatology at a tertiary care centre after approval from their institutional ethics committee. The study period was from November 2015 to January 2017.

In our study, sixty patients of AV were included. Inclusion criteria included untreated (washout period for topical and systemic therapy for acne: 2 and 4 weeks, respectively) cases of AV of age 15–45 years of either sex. The exclusion criteria included acne with the underlying polycystic ovarian syndrome (PCOS), metabolic syndrome, insulin resistance syndrome, pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome, synovitis, acne, pustulosis, hyperostosis and osteitis (SAPHO) syndrome, hyperandrogenism, autoimmune diseases and pregnant and lactating women. Furthermore, those with body mass index >25, history of type 1 diabetes mellitus or thyroid abnormalities and patients on drugs likely to cause acneiform eruptions were also excluded. Thirty age- and sex-matched volunteers were enrolled as controls.

PCOS, metabolic syndrome and insulin resistance syndrome were ruled out through history and clinical examination. The patient pro forma used for the assessment included the following:

1. Body mass index (BMI) >25

2. Waist circumference

3. Blood pressure

4. Menstrual history (in case of females)

5. Presence of Acanthosis nigricans

6. Multiple acrochordons

7. Xanthelasma palpebrarum

8. Hirsutism

9. Seborrhoeic dermatitis

Before enrolment, well-informed written consent was taken from each participant. A detailed history and clinical examination were performed for each patient. Patients were divided into five groups depending on the severity of disease: none (0), mild (1–18), moderate (19–30), severe (31–38) and very severe (>39) acne as per Global Acne Grading System (GAGS).[6]

Ten millilitres of fasting peripheral venous blood was collected in plain vacutainer tubes from cases and controls. The blood sample was allowed to stand for 30 minutes at room temperature and then centrifuged for 5 minutes at 2800 rpm. The serum was separated and stored at -20°C. Sandwich enzyme-linked immunosorbent assay (ELISA) is used to detect serum levels of IL-6, IL-8, IL-17 and IL-22 using different diagnostic kits of Krishgen Biosystems (USA), Diaclone (France), Diaclone (France) and Boster Biological Technology (USA), respectively, as per manufacturers’ instructions. The absorbance was read on a spectrophotometer using 450 nm as the primary wavelength.

Statistical analysis was performed using the statistical software Statistical Package for the Social Sciences (SPSS) version 21.0. Categorical variables were presented in number and percentage (%), and continuous variables were presented as mean ± SD and median. The normality of data was tested by the Kolmogorov–Smirnov test. The results were analysed using the unpaired t-test/Mann–Whitney test and analysis of variance (ANOVA)/Kruskal–Wallis test as appropriate. Qualitative variables were correlated using the Chi-square test/Fisher's exact test. A probability value of P < 0.05 was considered statistically significant.

Results

The mean age of cases was 22.35 years (SD = 5.31 years). The mean age of control was 21.86 years (SD = 3.78 years). The study group consisted of 39 (65%) females and 21 (35%) males. The control group included 20 (66.67%) females and 10 (33.33%) males. The groups were comparable as per age and gender distribution (P value: 0.898 and P value: 0.875, respectively). The mean duration of disease was 6.38 years (range: 1 to 15 years). Twenty-four (40%) patients had mild grade, whereas 31 (51.67%) and five (8.33%) had moderate and severe grades of acne, respectively.

Serum levels of IL-17 and IL-8 were significantly higher in cases (0.19 ± 0.0075 pg/ml and 0.38 ± 0.080 pg/ml) than in controls (0.13 ± 0.0033 pg/ml and 0.14 ± 0.034 pg/ml) (P < 0.0001 and P < 0.0001, respectively), while IL-6 and IL-22 were insignificantly higher in cases (cases = 0.15 ± 0.0174 pg/ml and 0.23 ± 0.0152 pg/ml, control = 0.13 ± 0.0095 pg/ml and 0.21 ± 0.0099 pg/ml) (P = 0.844 and P = 0.706, respectively) [Table 1]. Furthermore, IL-6 and IL-8 levels showed a significant positive correlation with increasing severity of acne [Table 2]. Also, IL-8 and IL-17 have a significant positive correlation (P < 0.0001).

Table 1.

Comparison of serum IL-6, IL-8, IL-17 and IL-22 levels between acne cases and controls

IL-6	Cases	Controls	P
Sample size	60	30
Mean±SE	0.15±0.0174	0.13±0.0095
Median	0.12	0.12	0.844
Min-max	0.06-0.83	0.04-0.27
Inter-quartile range	0.0945-0.153	0.103-0.143
IL-8
Sample size	60	30
Mean±SE	0.38±0.080	0.14±0.034
Median	0.13	0.09	<0.0001
Min-max	0.08-2.89	0.08-0.89
Inter-quartile range	0.106-0.246	0.084-0.098
IL-17
Sample size	60	30
Mean±SE	0.19±0.0075	0.13±0.0033
Median	0.19	0.13	<0.0001
Min-max	0.11-0.33	0.08-0.18
Inter-quartile range	0.146-0.235	0.119-0.136
IL-22
Sample size	60	30
Mean±SE	0.23±0.0152	0.21±0.0099	0.706
Median	0.19	0.19
Min-max	0.16-0.82	0.16-0.4
Inter-quartile range	0.174-0.221	0.180-0.204

Table 2.

Comparison between different interleukin levels with severity of acne

IL-6	Grades of acne			P	R
	Mild	Moderate	Severe
Sample size	24	31	5
Mean±SE	0.13±0.0294	0.16±0.0239	0.17±0.0477
Median	0.1	0.13	0.12	0.036	0.328
Min-max	0.06-0.79	0.07-0.83	0.1-0.35
Inter-quartile range	0.076-0.144	0.100-0.179	0.103-0.231
IL-8	Mild	Moderate	Severe
Sample size	24	31	5
Mean±SE	0.17±0.0323	0.3±0.0772	1.81±0.5082
Median	0.12	0.14	1.81	0.036	0.257
Min-max	0.08-0.83	0.08-1.94	0.1-2.89
Inter-quartile range	0.105-0.155	0.106-0.254	1.125-2.814
IL-17	Mild	Moderate	Severe
Sample size	24	31	5
Mean±SE	0.19±0.0115	0.19±0.0114	0.2±0.0206
Median	0.18	0.19	0.22	0.802	0.015
Min-max	0.13-0.33	0.11-0.33	0.13-0.25
Inter-quartile range	0.151-0.214	0.138-0.244	0.178-0.236
IL-22	Mild	Moderate	Severe
Sample size	24	31	5
Mean±SE	0.21±0.0153	0.24±0.0266	0.2±0.0185
Median	0.19	0.19	0.17	0.575	0.024
Min-max	0.16-0.49	0.16-0.82	0.17-0.27
Inter-quartile range	0.173-0.216	0.179-0.234	0.170-0.214

Discussion

The precise sequence of events in etiopathogenesis of AV remains unclear. The pilosebaceous unit is an immunocompetent organ in which keratinocytes and sebocytes act as immune cells.[7] These cells are capable of pathogen recognition and abnormal lipid formation. Previous studies have established IL-1α as the key cytokine in the pathogenesis of AV, mainly for the formation of microcomedones.[8] IL-1α upregulates the vascular cell adhesion molecules 1 (VCAM-1), intercellular cell adhesion molecules 1 (ICAM-1) and E-selectin expression and cellular components (CD4+ T cells and macrophages) and increases the proliferation of keratinocytes in the pilosebaceous unit.[9]

Current evidence suggests that inflammation sets in early in the course of acne pathogenesis, mainly triggered by TLR-2-mediated C. acnes recognition by keratinocytes and sebocytes.[10] Furthermore, CD4+ T cells and CD68+ macrophages have been observed at the site of early lesions, even before follicular hyper-cornification and comedone formation.[9] Our data showed significantly raised IL-8 and IL-17 levels in acne cases as compared to controls. IL-8 is a potent neutrophil chemotactic factor and is released by different cells, such as monocytes, macrophages and keratinocytes.[6] Degranulation of neutrophils at the site of inflammation leads to the release of proteolytic enzymes and reactive oxygen species (ROS), thus forming inflammatory lesions.[11] Neutrophils are present in pustular lesions of acne in abundance, and their levels show a correlation with the severity of acne.[12] IL-8 (along with IL-1β and TNF-α) is also released following C. acnes stimulation of monocytes via TLR-2.[13] Kim et al. observed the upregulation of TLR-2 proteins with the evolution of acne lesions leading subsequently to an increase in IL-8 levels. Hussain et al. reported IL-8-251T > A gene polymorphisms to be associated with circulating IL-8 levels and greater severity of acne.[11] Similar to our study, Zhog-Yong et al. and Anwar et al. reported the raised level of IL-8 with increasing severity of acne.[14,15] We also found serum level of IL-8 to correlate positively with the grading of acne (R = 0.257, P = 0.036). Furthermore, Sahib et al. also reported the raised level of IL-8 in acne patients compared with healthy volunteers.[16] Furthermore, Stankowska et al. evaluated serum IL-6 and IL-8 levels in acne patients and controls.[17] They reported raised IL-6 level, but IL-8 level showed no differences among the groups. They started as the severity of disease progresses and IL-6 levels increase. A further stimulant to neutrophil recruitment and subsequent inflammation in acne is the Th17 cells. We observed a significant positive correlation between the two cytokines, suggesting the Th17 cells may be the major producers of this cytokine in acne. Evidence has been recently building on the role of this cell line in acne pathogenesis.[12]

C. acnes has been shown to cause differentiation of naïve CD4+ cells into Th17 cells under the influence of TGF-β, IL-6, IL-1β and IL-23.[5] Also, Th17 pathway-activating cytokines such as IL-1β and IL-6 have been observed in acne lesions. Stimulated Th17 cells release cytokines such as IL-17A to IL-17F and IL-22.[5] IL-17A and IL-17F are not only the key cytokines leading to the recruitment and activation of neutrophils, but they can also target other cell types including keratinocytes, endothelial cells, monocytes and fibroblasts and stimulate the production of pro-inflammatory mediators—IL-6, TNF, IL-1β, PGE2 and matrix metalloproteinases leading to further damage at lesional sites.[18] Abd-Elmaged and Ebrahim reported significantly increased serum and tissue levels of IL-17 levels with increasing severity of acne.[19] In a similar study, Sinta et al. measured serum IL-17 level in acne patients and reported increased levels of IL-17 with increasing severity of acne.[20] Singh A et al. showed increased levels of IL-17 in acne patients as compared to controls. They also reported increased serum IL-17 levels with increasing severity of disease.[21] More recently, Yang et al. reported higher Th17 cell expression in lesional acne lesions (less than 48-hour-old) compared with nonlesional skin, via integrated bioinformatic assay.[22] Kistowska et al. demonstrated C. acnes-specific Th17 and Th17/Th1 cells in the peripheral blood of patients with acne lending support to the role of C. acnes in driving the Th17 inflammatory response.[23] Our study is in agreement with the previous literature as the serum level of IL-17 was significantly raised among cases than in controls but we found no significant change in the cytokine with increasing severity of acne (R = 0.015, P = 0.802). Also, in contrast to some in vitro studies performed previously, we did not observe higher IL-6 in cases compared with controls.[1,23]

Lesional release and concentration of the cytokine could be one cause for the discrepant results, but the assumption would require simultaneous measurement of skin and serum levels to be proven.

We observed only a small insignificant difference in IL-22 levels between cases and controls. This is similar to observations of Kistowska et al., who did not find any difference in IL-22 mRNA levels in skin biopsies of acne lesions as compared to normal skin, but in contrast to the in vitro study by Agak et al.[5,23] The former study reported a lack of induction of secretion of IL-4 and IL-22 from peripheral blood mononuclear cells of acne patients following stimulation with C. acnes, although IL-17 secretion was induced.

Th22 cell line has not been independently studied in AV as yet. It is likely that C. acnes stimulated differentiation of CD4+ T cells leads to IL-17-secreting Th17 cells with insignificant IL-22 expression. IL-22 functions synergistically with IL-17 or TNF. However, it plays different roles by IL-22/IL-22 receptor signal transductions in pathologic processes, including inflammation, autoimmunity, tumour and digestive organ damages.[24] In this regard, the plasticity of Th17 cell cytokine secretion has been demonstrated with the existence of IL-10+/IL-17+ Th17 cells and INF-γ+/IL-17+ Th17 cells in acne lesions. The effect of cutaneous microbiome in determining this plasticity and modulation of cytokine secretion has been highlighted by some authors recently.[5,25]

Thus, while the function of Th17 cell line and specifically IL-17 is clear, the intricacies of interaction of C. acnes with the adaptive immune system and resultant cytokine milieu are just beginning to be understood. Furthermore, with the current knowledge gaps, there is a long way before cytokine-specific treatments can be considered for clinical use in AV. However, the efficacy of existing drugs is being increasingly understood with respect to the effects on inflammatory pathways in the light of recent studies. This includes retinoic acid derivatives, which likely downregulate the Th17 pathway, and vitamins A and D, which have been shown in vitro to reduce C. acnes-induced Th17 differentiation.[1,26] Based on the existing literature and our findings, we believe that drugs modulating the microbe-inflammatory pathway interaction are more likely to show specific effects on AV, and our work and that of others in the domain lay the groundwork for future research on this aspect.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.