Correlation of Serum 25-Hydroxy Vitamin D and Interleukin-17 Levels with Disease Severity in Acne Vulgaris.

BACKGROUND: The association of Vitamin D (vit.D) and Interleukin 17 (IL-17) with acne vulgaris is uncertain in spite of induction of IL-17 by Propionibacterium acnes (P. acnes) and the role of vit.D in various inflammatory skin disorders including acne. The objectives of present study were to evaluate the levels of serum 25-hydroxyvitamin D3 [25(OH)D] and IL-17 in acne patients and age- and sex-matched controls and to compare them with the severity of acne as measured by Global Acne Grading System (GAGS).
METHODS: The study included 50 patients of acne and 30 healthy controls. Serum 25(OH) D and IL-17 levels were measured using chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA), respectively.
RESULTS: Vitamin D deficiency was detected in 28% of patients with acne but only in 6.7% of the healthy controls (P value 0.022). The levels of 25(OH)D were inversely associated with the severity of acne (P < 0.001). The mean serum IL-17 levels were significantly raised (P < 0.001) in acne patients (8.215 ± 5.33 pg/mL) as compared to controls (2.486 ± 2.12 pg/mL). A significant rise in levels of IL-17 was observed with the severity of acne (P < 0.001). Further, a highly significant negative correlation (Correlation Coefficient: -0.668) was noted between serum IL-17 and 25(OH) D levels along with disease severity in acne patients (P value < 0.001).
CONCLUSIONS: Raised IL-17 levels in acne correlate negatively with vit.D deficiency and both are significantly more prevalent in patients with acne as compared to healthy controls. Copyright:

Introduction

Acne vulgaris, seen in 85% of adolescents worldwide, is a chronic inflammatory disease of pilosebaceous units and manifests with polymorphic lesions such as closed and open comedones, papules, pustules, and nodules.[1] The various factors implicated in the acne pathogenesis include hormones, follicular plugging and hyperkeratinization, increased sebum secretion, P. acnes colonization and inflammation. The exact sequence of the events and their interconnection is, however, not well understood yet.

Vitamin D is known to regulate the immune system and the proliferation and differentiation of keratinocytes and sebocytes. It also has anti-comedogenic and antioxidant properties facilitating the synthesis of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).[2] The levels of these antioxidants are decreased in papulopustular acne.[3] Further, antimicrobial peptides like cathelicidins are induced by vit.D as reported by Liu et al.,[4] thereby reiterating the role of vit.D, in the etiopathogenesis of acne.

The immunological pathways involved in the pathogenesis of acne include T helper (Th) 1, Th2, and Th17 cells.[567] A recent pioneering work from the Kim lab by Agak et al.[8] on IL-17 has demonstrated that acne is a Th17-mediated disease. Kistowska et al.[9] also emphasized the role of Th17 and Th1 cytokines in acne vulgaris. A significant expression of the main effector cytokine of Th17 pathway (IL-17A) in acne lesions at mRNA and protein level has been documented by Kelhala et al.[10] in 2014, and in addition, they also found increased levels of Th17 promoter cytokines (IL-1β, IL-6, TGF-β, and IL-23) at mRNA level.

Joshi et al.[11] reported that 1,25 dihydroxy vitamin D 1,25(OH) 2D] inhibits IL-17 production by Th17 cells via direct transcriptional suppression of IL-17 gene expression. Vitamin D also inhibits P. acnes-induced Th17 differentiation and thereby could be an effective tool in modulating acne.[8]

Till date, only a few studies have assessed the relationship between vit.D and IL-17 levels in acne vulgaris, though the two factors have individually been implicated in its pathogenesis.[81213] In the present study, we have compared serum levels of vit.D and IL-17 in acne patients with healthy controls.

Materials and Methods

This cross-sectional observational study was conducted in the department of dermatology at a tertiary care hospital in Delhi, India after approval from the institutional ethics committee. The study group comprised of 50 newly diagnosed, untreated, consenting acne patients (age 15--45 years) with varying grades of severity. Patients with features of hyperandrogenism, polycystic ovarian syndrome, metabolic syndrome, insulin resistance, or any autoimmune disease were excluded. Patients with history of intake of drugs which were likely to cause acneiform eruptions, or taking vit.D supplements, oral steroids, and alcohol were also excluded from the study. Acne severity was objectively assessed using the Global Acne Grading System (GAGS).[14] Thirty, age- and sex-matched consenting healthy volunteers belonging to the same geographic region were enrolled as controls. Baseline venous blood samples (5 ml) were collected from patients and control groups under aseptic conditions. Serum was separated after centrifugation and were stored at -80°C until the estimation of serum 25(OH) D and IL-17.[15]

Serum 25(OH)D was estimated by enhanced CLIA using kits by Eci VITROS orthoclinical diagnostics, India. The levels of 25(OH) D were categorized as adequate (>30 ng/mL), insufficient (10–30 ng/mL), and deficient (<10 ng/mL). The concentration of serum IL-17A was measured quantitatively in the preserved serum samples by the ELISA method using a kit from Diaclone, France.[1617]

All data were analyzed by statistical software SPSS 20.0.0. (SPSS Inc. Chicago, IL, U.S.A.). Two sided P values less than 0.05 were considered statistically significant. Continuous variables were summarized as mean ± standard deviation (SD). Categorical variables were expressed as number (percentage). The Shapiro--Wilk's W-test was applied for checking the normality assumption of continuous variables. Chi-square test and Fisher's exact test, wherever appropriate, were used for data analysis. Post-hoc (Bonferroni) test was used for comparison of mean 25(OH)D and IL-17 levels among various grades of acne. The correlation between acne severity (GAGS score) and other parameters was assessed using Spearman's correlation test.

Results

Patient and control groups were comparable with respect to age, sex, and body mass index [Table 1]. The mean serum 25(OH) D level was lower in acne patients (16.652 ± 8.63 ng/ml) than in controls (20.904 ± 11.66 ng/ml), although the difference was not statistically significant, however, the prevalence of vit.D deficiency (serum 25(OH)D <10 ng/ml) was significantly higher in patients with acne as compared to controls (28% vs. 6.7%; P value 0.022). Further, gender-based comparison of serum 25(OH)D levels between cases and controls also did not reveal any statistically significant difference {males (P 0.087) and females (P 0.344)}.

Table 1

Baseline demographic and clinical characteristics of patients with acne and controls

	Acne patients	Controls	P
Age (year)	22.44±3.073	24±4.152	0.059
Sex (F/M)	11/39 (22/78)	9/21 (30/70)	0.423
BMI (kg/m2)	23.678±2.998	22.417±2.394	0.057
Serum 25(OH)D levels (ng/mL)	16.652±8.63	20.904±11.66	0.066
Male	16.890±8.71	21.402±11.11	0.087
Female	15.810±8.69	19.742±9.39	0.344
Vitamin D deficiency	28%	6.7%	0.022
IL-17 levels (pg/mL)	8.215±5.33	2.486±2.12	<0.001

The mean 25(OH)D value was 28.06 ng/ml in mild grade acne, 21.63 ng/ml in moderate acne, 13.34 ng/ml in severe grade, and 9.47 ng/ml in very severe grade of acne. There was a significant negative correlation between vit.D levels and severity of acne based on GAGS (P < 0.001) [Table 2]. Also, 9 of the 15 patients (60%) in the very severe grade and 5 out of 15 patients (33.3%) of severe acne were vit.D deficient, whereas none of the 20 patients in the mild and moderate grade of acne had vit.D deficiency (P value < 0.001). Post-hoc (Bonferroni) test for the comparison of mean 25(OH) D levels [Table 3] among various grade of acne revealed that there was a significant difference in the mean levels of 25(OH) D in cases of very severe and severe grades in comparison to mild and moderate grades of acne. Interestingly, no significant difference in mean 25(OH) D levels was found between mild and moderate grade acne as well as between severe and very severe grades of acne. Vitamin D deficiency was significantly more prevalent in vegetarian patients (43.33%) as compared to 5% of non-vegetarian acne patients (P value 0.004). Further, we noted a significantly higher prevalence (P value <0.001) of vit.D deficiency in patients with truncal lesions (46.4%) as compared to individuals without truncal lesions (4.5%). No significant correlation was seen between deficient serum 25(OH) D levels and age, gender, family history, skin type, age of onset and disease duration of acne.

Table 2

Mean IL-17 and serum 25(OH) D levels in acne cases with respect to acne grade and truncal lesions

	Mean IL-17 levels (pg/mL)	P	Mean 25(OH) D levels (ng/mL)	P
Acne Grade (n)
Mild (9)	2.948±2.04	<0.001	28.06±9.66	<0.001
Moderate (11)	4.881±2.38		21.63±4.84
Severe (15)	8.504±4.03		13.34±4.26
Very severe (15)	13.533±4.13		9.47±1.5
Truncal lesions (n)
Absent (22)	4.705±3.54	<0.001	22.879±8.84	<0.001
Present (28)	10.974±4.89		11.760±4.23

Table 3

Post hoc (Bonferroni) test for comparison of mean serum 25(OH) D levels and IL-17 levels between various grades of acne

Groups	Difference between mean 25(oh) d levels (ng/ml)	P	Difference between mean il-17 levels (pg/ml)	P
Mild grade
Moderate grade	6.43	0.055	1.94	1.000
Severe grade	14.72	<0.001	7.56	0.004
Very severe grade	18.59	<0.001	10.59	<0.001
Moderate grade
Severe grade	8.29	0.001	3.62	0.089
Very severe grade	11.16	<0.001	8.65	<0.001
Severe grade
Very severe	3.87	0.296	5.03	0.002
grade

Mean serum IL-17 levels were significantly higher (P < 0.001) in cases (8.215 ± 5.33 pg/ml) than controls (2.486 ± 2.12 pg/ml). Mean serum IL-17 level was 2.948 pg/ml in mild acne, 4.881 pg/ml in moderate grade, 8.504 pg/ml in severe grade, and 13.533 pg/ml in very severe acne [Table 2]. The rise in levels of IL-17 with increasing severity of acne was highly significant (P < 0.001). Post-hoc (Bonferroni) test for comparison of mean IL-17 levels [Table 3] between various grades of acne showed that there was a significant difference in the mean levels of IL-17 in very severe grade of acne as compared to mild, moderate, and severe grades, whereas there was no significant difference in mean IL-17 levels between mild and moderate grade acne or between moderate and severe grades of acne. Mean IL-17 level in cases with truncal lesions (10.974 ± 4.89 pg/ml) were significantly higher (P value < 0.001) than mean IL-17 level in patients without truncal lesions (4.705 ± 3.54 pg/ml). No significant correlation was seen between raised serum IL-17 levels and age, gender, skin type, age of onset of acne, disease duration, dietary pattern, and family history of acne.

Interestingly, raised serum IL-17 levels (> 3 pg/ml) were seen in 92.8%, 78.1%, and 25% of the patients with vit.D deficiency, insufficiency, and normal 25(OH) D levels, respectively (P value 0.029). Further, there was a significant (P < 0.001) negative correlation between serum levels of 25(OH) D and IL-17 in acne patients (correlation coefficient = -0.668) [Figure 1].

Figure 1

Scatter diagram showing the variation of serum IL-17 levels with respect to serum 25(OH)D levels

Discussion

The skin is not only the source of vit.D for the body but is also capable of responding to the active metabolite of vit.D, that is, 1,25(OH) 2D. The key components of the vit.D system (vit.D receptor, 25-hydroxylase, 1α-hydroxylase, and 24-hydroxylase) are strongly expressed in sebocytes.[18] Both calcium and 1,25(OH) 2D perform interacting functions in regulating the skin differentiation process. Vitamin D can stimulate or inhibit keratinocyte proliferation at lower or higher concentrations (≥10-8 M), respectively.[19] The biphasic effect of 1,25(OH) D on sebocytes causes inhibition of rapidly proliferating and stimulation of slowly proliferating Z95 sebocytes as documented by Krämer et al.[18] Besides, the rise of inflammatory cytokines (IL6, IL8 and MMP9) has been reported from cultured sebocytes in vit.D deficiency.[20]

Vitamin D has known immunomodulatory effects on T and B lymphocytes, dendritic cells, Toll- like receptor 2 (TLR2) and its co-receptor CD14, and is known to upregulate the innate immune response in skin.[21] vit.D also induces Forkhead box O (FoxO) deacetylation and dephosphorylation resulting in its activation, further leading to inhibition of hepatic IGF-1 secretion.[22] vit.D also activates FoxO signaling which strongly inhibits mammalian target of rapamycin complex 1 (mTORC1), thereby inhibiting signaling through IGF-1, a major pathway in acne pathogenesis.[23]

Recently various cytokines including IL-17 have been implicated in acne pathogenesis. Vitamin A (ATRA) and 1,25(OH) 2D downregulate the expression of IL-17 at mRNA and protein level and also inhibit P. acnes mediated Th17 differentiation because of the common signaling pathway involving retinoid X receptor. Interestingly, a combination of ATRA and 1,25(OH) 2D inhibited retinoid orphan receptors α (RORα) and RORγ expression more strongly, thereby suggesting synergistic action of both on retinoid receptors.[8]

There is a paucity of studies that have assessed vit.D status along with the evaluation of serum IL-17 levels in acne patients. The present study did not find any significant differences in the mean 25(OH) D levels between acne patients and controls. This could be because of the widely prevalent vit.D deficiency in the Indian population per se. Further, sex-based comparison of serum 25(OH) D levels between patients and controls also did not reveal any significant difference. However, the prevalence of vit.D deficiency was significantly higher in patients with acne (28%) compared to healthy controls (6.7%). Similar findings have been previously reported by Lim et al.,[24] El Hamd et al.,[25] and Abu-Elmaged et al.[12] However, studies by Toossi et al.[26] and Al-Taiar et al.[27] did not find any significant association between acne vulgaris and serum 25(OH)D levels as well as no correlation with severity of acne which the researchers explained could be because of low serum vit.D levels among Iranian and Kuwaiti population, respectively.

Our study also revealed a significant negative correlation of vit.D with acne severity, presence of truncal lesions, and dietary pattern (vegetarian diet) of the patients. Similarly, inverse correlation of vit.D deficiency with increasing severity of acne has also been reported in various studies.[122425] In our study, most of the patients with truncal lesions had severe grades (GAGS >30) of acne and were vit.D deficient.

We observed a highly significant (P < 0.001) difference in mean serum levels of IL-17 between acne patients and controls. Some previous reports have shown raised tissue IL-17 levels in acne patients, while few recent studies from Egypt have shown increased intralesional as well as serum IL-17 levels.[712] The higher serum IL-17 levels are likely due to a spill-over of increased IL-17 secretion at the disease site. The high levels in serum resulting from a disease localized to a small area of skin is reflective of the quantum of this cytokine produced at one site and emphasizes its important role in the pathogenesis of acne. Further, we noted a significant rise in IL-17 levels with increasing grade of acne which was in concordance with results of Murlistyarini et al.[28] and Ebrahim et al.[7] lending further support to the association. However, this finding was contrary to the findings of Maulinda et al.[29] and Topan et al.[13] who did not find any significant difference in serum IL-17 levels between patients of papulopustular and comedonal acne. Further in our study, acne patients with truncal lesions had significantly higher mean levels of IL-17 which is indicative of the severity of acne at this site. This probably signifies the predominant role of IL-17 in the inflammatory stages or progression of acne.

The evidence of Th17 lineage involvement in acne has been provided by studies documenting raised levels of IL-17A and IL-21 in acne lesions and the presence of P.acnes induced Th17 and Th17/Th1 cells in peripheral blood of acne patients.[8] Also, higher IL-17 levels are induced by P. acnes strain isolated from acne lesions (PA) than healthy skin (PH).[30] Further, Matti et al.[31] documented that sebocytes functionally interact with P. acnes in inducing maturation of dendritic cells which result in preferential priming of Th17 cells in response to P.acnes.

Our study also showed significantly higher IL-17 levels in acne patients with vit.D deficiency as compared to patients with normal vit.D levels (P value 0.029). There was a significant decrease in the mean serum 25(OH)D levels in very severe and severe grades as compared to mild and moderate grades of acne along with a significant increase in the mean levels of IL-17 in cases of the very severe grade of acne in comparison to mild, moderate, and severe grades. This is in line with the documented immunomodulatory effect of vit.D in regulating the Th17 pathway. A similar negative correlation between serum 25(OH)D and serum IL-17 levels has been documented by Abd-Elmaged et al.,[12] however, they found increased IL-17 levels even in the mild stage of acne while in our study, raised IL-17 levels were seen in severe grades of acne. A recent study from Turkey did not find any correlation between serum 25(OH)D levels and IL-17 with acne.[13] Similar findings of decreased serum 25(OH)D levels and increased serum IL-17 levels have been reported in other chronic inflammatory skin diseases like vitiligo and psoriasis.[3233] However, there was no significant negative correlation between vit.D and IL-17 in these diseases as has been reported in our study.

Our findings have therapeutic implications since drugs acting on the Th17 pathway such as dihydroxy vitamin D3, retinoids, and zinc have been reported to be effective in the treatment of acne and future research for drugs inhibiting Th17 pathway may be crucial.[2534] A randomized controlled trial with vit.D supplementation in acne patients for 2 months resulted in a statistically significant improvement in the inflammatory lesions in the vitamin D group compared with the control group.[24] Conversely, the treatment of acne with retinoids has shown to improve serum vit.D levels in patients.[7] However, the findings need confirmation with larger patient numbers and in varied racial groups.

Conclusion

The present study revealed that serum 25(OH) D levels inversely correlate with IL-17 levels, in increasing grades of severity of acne. The evaluation of vit.D levels and its therapeutic intervention must be explored in all acne cases, more so in patients with severe grades of acne, truncal lesions, and on a vegetarian diet. Further studies evaluating the tissue levels of vit.D in acne patients are also required to reveal direct evidence of the effect of vit.D on acne. In light of our findings and the literature on increased expression of IL-17 in acne lesions along with other factors involved in the pathogenesis of acne, a sui generis approach for the treatment of acne needs to be explored so as to target multiple interlinked events in acne.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.