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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 2  |  Page : 147-150

Increased expression of interleukin-17A in the lesional skin indicates increase of serum antibody anti-phenolic glycolipid-I in leprosy patients


Department of Dermatology and Venereology, Faculty of Medicine, Universitas Padjadjaran-Dr. Hasan Sadikin Hospital, Bandung, West Java, Indonesia

Date of Web Publication23-Nov-2017

Correspondence Address:
Hendra Gunawan
Department of Dermatology and Venereology, Faculty of Medicine, Universitas Padjadjaran. Dr. Hasan Sadikin Hospital, Jl. Pasteur No. 38, Bandung, West Java 40161
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_84_17

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  Abstract 


Background: In lepromatous type of leprosy, the serum titer of the antibody against phenolic glycolipid (PGL)-I-a-specific antigen in leprosy that is produced by B cell is high. Whereas, the interleukin (IL)-17A expressed by T-helper 17 cells could induce B-cell differentiation. The role of IL-17A in leprosy is still unknown. Hence, this study aimed to assess a correlation between IL-17A expression in the lesional skin and anti-PGL-I immunoglobulin (Ig) M serum levels in leprosy patients. Methods: This study was performed in Leprosy Clinic, Dr. Hasan Sadikin Hospital Bandung, Indonesia, using a cross-sectional analytical study. A punch biopsy obtained from 49 leprosy patients was included through consecutive sampling for measurement of IL-17A expression in the skin by immunohistochemistry scoring (histoscore). Furthermore, the anti-PGL-I IgM level in serum is evaluated by enzyme-linked immunosorbent assay. Results: The IL-17A expressions in the skin biopsies of tuberculoid (TT), borderline tuberculoid (BT), mid-borderline (BB), borderline lepromatous (BL), and lepromatous leprosy (LL) patients using histoscore were 1.00, 2.31, 4.63, 5.06, and 10.14, respectively, and they showed significant differences (P = 0.0001). The titer of anti-PGL-I IgM levels was 89 pg/ml, 555 pg/ml, 1.244 pg/ml, 1.920 pg/ml, and 23.591 pg/ml in TT, BT, BB, BL, and LL patients, respectively, and they showed significant differences (P = 0.005). The results of Rank–Spearman correlation analysis between IL-17A expression in the skin and anti-PGL-I IgM serum levels were as follows: r = 0.767 and P = 0.0001. Conclusion: These results suggested that the increase of IL-17A expression in the lesional skin indicates the increase of anti-PGL-I IgM serum levels in leprosy.

Keywords: Anti-phenolic glycolipid-I immunoglobulin M, interleukin-17A, leprosy


How to cite this article:
Gunawan H, Annissa MN, Dwiyana RF, Avriyanti E, Suwarsa O. Increased expression of interleukin-17A in the lesional skin indicates increase of serum antibody anti-phenolic glycolipid-I in leprosy patients. Biomed Biotechnol Res J 2017;1:147-50

How to cite this URL:
Gunawan H, Annissa MN, Dwiyana RF, Avriyanti E, Suwarsa O. Increased expression of interleukin-17A in the lesional skin indicates increase of serum antibody anti-phenolic glycolipid-I in leprosy patients. Biomed Biotechnol Res J [serial online] 2017 [cited 2019 Mar 21];1:147-50. Available from: http://www.bmbtrj.org/text.asp?2017/1/2/147/219115




  Introduction Top


Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae,[1],[2] which mainly affects the peripheral nerves and the skin.[1],[2],[3] The important diversity of clinical and pathological findings related to leprosy is a result of the variable levels of cellular and humoral immunity to M. leprae among the patients affected by it.[3],[4] The abnormality of CD + T-lymphocyte immunological function is the most important pathogenesis in leprosy. The T-helper (Th) 17 cell, a new kind of CD4 + T-cell subpopulation found recently, highly produces interleukin (IL)-17 or IL-17A.[5] The presence of IL-17A mRNA in the skin of lepromatous patients but not in tuberculoid (TT) type was performed by Cirée et al.[6] However, the role of IL-17A in leprosy is still unknown.[7]

Lepromatous type of leprosy is known for high anti-phenolic glycolipid (PGL)-I immunoglobulin (Ig) M serum levels[8] and the M. leprae- specific antigen.[1],[2],[3] This anti-PGL-I IgM is produced by B cells,[9] while IL-17A also expressed by Th17 cells that could induce B-cell differentiation.[10] Thus, we want to know the correlation of IL-17A expression in the skin and anti-PGL-I IgM in leprosy patients.


  Methods Top


Patients

This study was an observational analytical study within cross-sectional design. Forty-nine leprosy patients (9 females and 40 males; mean age 30 ± 16.8 years old) from Leprosy Clinic, Dr. Hasan Sadikin Hospital Bandung, Indonesia, were obtained through consecutive sampling.

Leprosy patients were grouped according to the clinical classification of Ridley and Jopling and consist of 1 TT, 16 borderline tuberculoid (BT), 8 mid-borderline (BB), 17 borderline lepromatous (BL), and 7 lepromatous leprosy (LL) patients. Blood sampling for anti-PGL-I IgM and IL-17A serum level assessment was performed in all subjects.

The study was approved by the Health Research Ethics Committee, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia. Oral explanation about the study was made, and subsequently written informed consent was obtained from each participant.

Interleukin-17A expression

IL-17A expression in the skin was examined by immunohistochemistry method. Briefly, skin biopsy specimens were obtained through 5-mm punch biopsy from each patient. Immunohistochemistry analysis was done using IL-17A antibody (US Biological, Massachusetts MA, USA) with 1:100 dilution. Expression of IL-17A in the skin was quantified using an immunohistochemistry score (histoscore) ([intensity + 1] × distribution) and assessed by a certified pathologist.

Enzyme-linked immunosorbent assay

Anti-phenolic glycolipid-I immunoglobulin M level

Anti-PGL-I IgM level was examined using enzyme-linked immunosorbent assay (ELISA) method.[11] An ELISA plate was coated with 50 μl of natural trisaccharide-phenyl-bovine serum albumin (0.431 nmol of sugar/ml) and then, the surface of the wells was blocked by incubation with 1% bovine serum albumin (BSA) at 37°C for 60 min. Serum was diluted (1:300) with dilution buffer (phosphate-buffered saline with 20% fetal calf serum and 0.5% Tween 20) and added to the wells and incubated at 37°C for 60 min. After the plate was washed with washing buffer (phosphate-buffered saline with 0.05% Tween 20), peroxidase-conjugated antihuman IgG or IgM antiserum (Dako Igs A/S, Copenhagen, Denmark) was added to the wells and incubated at 37°C for 60 min. The excess of conjugates was removed with washing buffer, and then, a substrate solution (0.4 mg of o-phenylenediamine/ml and 0.4 μl of 30% hydrogen peroxide/ml in 0.1 M citrate-phosphate buffer, pH 5.0) was added, and the plates were developed in the dark for 15–30 min. The reaction was stopped by 1.25 M sulfuric acid. The levels of anti-PGL-I IgM in pg/ml were measured by an ELISA plate reader (Biolise, Vermont, USA).

Interleukin-17A levels in serum

Serum levels of IL-17A were measured by an ELISA using commercially available kits for IL-17A (R & D System, Minneapolis, USA) in accordance with the manufacturer's instructions.

Statistical analysis

Data were expressed as mean. The Rank–Spearman test was used for correlation analysis. P values < 0.05 were considered statistically significant results.


  Results Top


Interleukin-17A expression in leprosy patients

IL-17A expression quantification in the skin was performed. It was found that IL-17A was expressed in 46 of 49 patients' skin (93.87%). It was mainly expressed in the epidermis (93.87%), papillary dermis (91.83%), reticular dermis (46.93%), and periadnexal region (87.75%) [Table 1] and [Figure 1]. We also found IL-17A expression in keratinocytes and granulomas in the skin of leprosy patients [Figure 2].
Table 1: Distribution of interleukin.17A skin expression in leprosy

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Figure 1: Immunohistochemistry staining of interleukin-17A expression in epidermis, papillary, and reticular dermis of leprosy patients' skin biopsy (magnification ×40)

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Figure 2: (a) Immunohistochemistry staining of interleukin-17A expression in keratinocytes and granulomas of leprosy patients' skin biopsy. (magnification ×40). The box shows inset. (b) A picture of inset in (a) in higher magnification

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Comparison of IL-17A expression in the skin between clinical types of leprosy revealed statistically significant differences. In contrary, IL-17A serum levels between clinical types of leprosy showed insignificant differences [Table 2].
Table 2: Interleukin-17A skin expression, interleukin-17A serum levels, and anti-phenolic glycolipid-I serum level among leprosy type

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The IL-17A expressions in skin biopsies of TT, BT, BB, BL, and LL type of leprosy using histoscore were 1.00, 2.31, 4.63, 5.06, and 10.14, respectively, and they showed significant differences (P = 0.0001).

Anti-phenolic glycolipid-I immunoglobulin M level

The titer of anti-PGL-I IgM levels was 89 pg/ml,; 555 pg/ml, 1.244 pg/ml, 1.920 pg/ml, and 23.591 pg/ml in TT, BT, BB, BL, and LL type of leprosy, respectively, and they showed significant differences (P = 0.005).

Correlation between interleukin-17A and anti-phenolic glycolipid-I immunoglobulin M

The correlation analysis results between IL-17A skin expression and anti-PGL-I IgM serum levels using Rank–Spearman test were as follows: r = 0.767 and P = 0.0001. This showed a very significant positive correlation [Figure 3]. We suggested that the increase of IL-17A expression in the skin indicated the increase of anti-PGL-I IgM serum in leprosy.
Figure 3: Significant positive correlation between interleukin-17A expression in the skin and anti-phenolic glycolipid-I immunoglobulin M mean levels in leprosy patients (r = 0.767; P = 0.0001) was obtained using Rank-Spearman test analysis

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  Discussion Top


IL-17A expression in the skin using immunohistochemical method in leprosy has never been reported before. To our knowledge, the present set of data provides the first evidence of IL-17A expression in the skin using immunohistochemistry method in leprosy patients.

Another analysis to examine IL-17A in leprosy has been described. Cirée et al.[6] showed the presence of IL-17A m-RNA through reverse transcriptase polymerase chain reaction method in LL skin, whereas no IL-17A mRNA could be detected in the TT leprosy skin lesions examined. da Motta-Passos et al.[12] also demonstrated the presence of IL-17A mRNA in leprosy patients' skin that was found to be higher in lepromatous type than tuberculoid type. Those results of Cirée and da Motta-Passos studies were similar to this study, that the expression of IL-17A in the skin was significantly higher in lepromatous than tuberculoid type of leprosy [Table 2]. The IL-17A was expressed in the epidermis, papillary dermis, reticular dermis, and periadnexal tissue [Table 1].

Leprosy is manifested by a broad spectrum of clinical and immunology, ranging from TT leprosy to LL. Th1 cells are predominant in TT type, whereas the Th2 cytokine pattern is more prominent in lepromatous type. IL-17A is produced by Th17[13] and also Th1, Th2,[14] or B cell.[9] In tuberculosis, IL-17A is inhibited by interferon-γ in vitro,[15] explaining the presence of higher IL-17A in lepromatous type than TT type of leprosy in our study.

Then, we compared the expression of IL-17A in the skin between types of leprosy using immunohistochemistry method. We found that IL-17A expression in the skin in each of clinical forms of leprosy showed significant differences. Therefore, IL-17A expression in the skin was higher in the lepromatous type compared with the TT type of leprosy. On the contrary, when we analyzed the IL-17A level in the serum between clinical forms of leprosy, it showed no significant differences [Table 2]. Taken together, we suggest that IL-17A expression in the skin represented the type of leprosy.

We also found that there were statistically significant differences of anti-PGL-I IgM mean levels in various types of leprosy patients, and its level increases in lepromatous type [Table 2]. Another group also mentions that anti-PGL-I IgM antibodies have been correlated to the total bacillary load of leprosy patients and have been reported to be adequate to monitor therapy, since antibody titer is decreasing with therapy.[16] Furthermore, we reported for the first time that IL-17A expression in the skin showed a very significant positive correlation with the anti-PGL-I IgM serum levels in leprosy patients. The main source of IL-17A in this study could be B cells, but further researches are needed.


  Conclusion Top


The increase of IL-17A expression in the lesional skin indicates the increase of anti-PGL-I IgM serum levels in leprosy. Hence, IL-17A could act as an adjunctive indicator of leprosy type and target of therapy in leprosy.

Financial support and sponsorship

This study was funded by Hibah Internal Universitas Padjadjaran, Bandung, West Java, Indonesia.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Rea TH, Modlin RL. Leprosy. In: Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, editors. Fitzpatrick's Dermatology in General Medicine. New York: McGraw Hill; 2008. p. 1786-96.  Back to cited text no. 1
    
2.
Lockwood DN. Leprosy. In: Burns T, Breatnach S, Cox N, Griffiths C, editors. Rook's Textbook of Dermatology. Oxford: Blackwell; 2004. p. 29.1-17.  Back to cited text no. 2
    
3.
Bryceson A, Pfaltzgraff RE. Leprosy. 3rd ed. London: Churchill Livingstone; 1990. p. 1-42.  Back to cited text no. 3
    
4.
Ottenhoff TH. New insights and tools to combat leprosy nerve damage. Lepr Rev 2011;82:334-7.  Back to cited text no. 4
    
5.
Weaver CT, Harrington LE, Mangan PR, Gavrieli M, Murphy KM. Th17: An effector CD4 T cell lineage with regulatory T cell ties. Immunity 2006;24:677-88.  Back to cited text no. 5
    
6.
Cirée A, Michel L, Camilleri-Bröet S, Jean Louis F, Oster M, Flageul B, et al. Expression and activity of IL-17 in cutaneous T-cell lymphomas (mycosis fungoides and Sezary syndrome). Int J Cancer 2004;112:113-20.  Back to cited text no. 6
    
7.
Yamamura M, Uyemura K, Deans RJ, Weinberg K, Rea TH, Bloom BR, et al. Defining protective responses to pathogens: Cytokine profiles in leprosy lesions. Science 1991;254:277-9.  Back to cited text no. 7
    
8.
Koster FT, Scollard DM, Umland ET, Fishbein DB, Hanly WC, Brennan PJ, et al. Cellular and humoral immune response to a phenolic glycolipid antigen (PhenGL-I) in patients with leprosy. J Clin Microbiol 1987;25:551-6.  Back to cited text no. 8
    
9.
Vazquez-Tello A, Halwani R, Li R, Nadigel J, Bar-Or A, Mazer BD, et al. IL-17A and IL-17F expression in B lymphocytes. Int Arch Allergy Immunol 2012;157:406-16.  Back to cited text no. 9
    
10.
Mitsdoerffer M, Lee Y, Jäger A, Kim HJ, Korn T, Kolls JK, et al. Proinflammatory T helper type 17 cells are effective B-cell helpers. Proc Natl Acad Sci U S A 2010;107:14292-7.  Back to cited text no. 10
    
11.
Prakoeswa CR, Agusni I, Izumi S. Detection of Mycobacterium leprae DNA in blood of the subclinical leprosy. Folia Med Indones 2007;43:64-7.  Back to cited text no. 11
    
12.
da Motta-Passos I, Malheiro A, Gomes Naveca F, de Souza Passos LF, Ribeiro De Barros Cardoso C, da Graça Souza Cunha M, et al. Decreased RNA expression of interleukin 17A in skin of leprosy. Eur J Dermatol 2012;22:488-94.  Back to cited text no. 12
    
13.
Steinman L. A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat Med 2007;13:139-45.  Back to cited text no. 13
    
14.
Albanesi C, Scarponi C, Cavani A, Federici M, Nasorri F, Girolomoni G, et al. Interleukin-17 is produced by both Th1 and Th2 lymphocytes, and modulates interferon-gamma- and interleukin-4-induced activation of human keratinocytes. J Invest Dermatol 2000;115:81-7.  Back to cited text no. 14
    
15.
Scriba TJ, Kalsdorf B, Abrahams DA, Isaacs F, Hofmeister J, Black G, et al. Distinct, specific IL-17- and IL-22-producing CD4+T cell subsets contribute to the human anti-mycobacterial immune response. J Immunol 2008;180:1962-70.  Back to cited text no. 15
    
16.
Steinman L. A rush to judgment on Th17. J Exp Med 2008;205:1517-22.  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

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