J Infect Dis.-2012-Sousa-1417-24

M A J O R A R T I C L E

Genetic and Immunological Evidence ImplicatesInterleukin 6 as a Susceptibility Gene for LeprosyType 2 Reaction

Ana Lucia M. Sousa,1,a Vinicius M. Fava,2,a Lucas H. Sampaio,1 Celina Maria T. Martelli,1 Mauricio B. Costa,1

Marcelo T. Mira,2,b and Mariane M. A. Stefani1,b

1Tropical Pathology and Public Health Institute, Federal University of Goias, Goiania; and 2Core for Advanced Molecular Investigation, GraduateProgram in Health Sciences, Pontifcia Universidade Catolica do Parana, Curitiba, Parana, Brazil

In leprosy, type 1 reaction (T1R) and type 2 reaction (T2R) are major causes of nerve injury and permanent

disabilities. A previous study on plasma levels of 27 cytokines in patients with T1R or T2R and controls with

nonreactional leprosy identified the gene for interleukin 6 (IL-6) as a candidate for genetic analysis. Two nested

case-control studies were built from a cohort of 409 patients with leprosy from central Brazil, monitored for

T1R and T2R. There was evidence for association between T2R and IL-6 tag single-nucleotide polymorphisms

rs2069832 (P5 .002), rs2069840 (P5 .03), and rs2069845 (P5 .04), with information on the entire IL-6 locus,

as well as functional IL-6 variant rs1800795 (P 5 .005). Moreover, IL-6 plasma levels in patients with T2R

correlated with IL-6 genotypes (P5 .04). No association was found between IL-6 variants and T1R. Identifying

genetic predictive factors for leprosy reactions may have a major impact on preventive strategies.

Leprosy is a chronic, dermatoneurological, disabling

disease caused by Mycobacterium leprae that, in 2009,

affected approximately 245 000 individuals worldwide.

Prevalence of the disease has remained stable since the

mid-1990s despite global implementation of efficient

multidrug therapy (MDT) [1]. Leprosy presents as

a broad clinical, bacilloscopic, histopathological, and

immunological spectrum, ranging from the localized

tuberculoid (TT) form, associated with a granulomatous

T-helper 1 (Th1)type immune response, to systemic

T-helper 2 (Th2)type lepromatous (LL) disease. Im-

munologically unstable borderline forms (borderline

tuberculoid [BT], borderline borderline [BB], and

borderline lepromatous [BL]) lie between the poles [2].

Leprosy reactions are immune inflammatoryrelated

phenotypes that may occur before diagnosis, during

treatment, or after MDT. Leprosy reactions require

immediate treatment to prevent permanent nerve im-

pairment, motor disability, and deformity. Cohort

studies estimate that disability in leprosy ranges from

16% to 56% [3], mainly owing to the occurrence of

reactional episodes. Because leprosy reactions may occur

months or even years after MDT completion, related

disabilities are expected to continue to occur even under

the unlikely scenario of leprosy eradication.

There are 2 major types of leprosy reactions. Type 1

reaction (T1R) affects 20%30% of patients [4] and is

characterized by acute inflammation of preexisting

skin lesions or by the appearance of new lesions and/or

neuritis. Systemic symptoms are rare. Type 1 reaction

occurs predominantly among patients with leprosy

presenting unstable immune response against the bacilli

(BT, BB, BL forms) [5] or in patients with TT leprosy

[6, 7]. Approximately 95% of T1R cases are diagnosed

simultaneously with leprosy or during the first 2 years

of MDT [8]. Even with adequate treatment, 40% of

Received 10 May 2011; accepted 18 October 2011; electronically published 29March 2012.

aA. L. M. S. and V. M. F. contributed equally to the work.bM. T. M. and M. M. A. S. share senior authorship.Presented in part: 11th Brazilian Congress of Leprosy, Porto Alegre, Rio Grande

do Sul, Brazil, 25 November 2008.Correspondence: Mariane Stefani, PhD, Rua 235, Sala 335, IPTSP, Universidade

Federal de Goias, Setor Universitario, Goiania, GO 74605050, Brazil ([email protected]).

The Journal of Infectious Diseases 2012;205:141724 The Author 2012. Published by Oxford University Press on behalf of the InfectiousDiseases Society of America. All rights reserved. For Permissions, please e-mail:[email protected]: 10.1093/infdis/jis208

IL-6 Polymorphism and Leprosy Type 2 Reaction d JID 2012:205 (1 May) d 1417

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patients with T1R may present permanent nerve damage [9].

Erythema nodosum leprosum is the main presentation of type 2

reaction (T2R), which predominantly affects patients with lep-

rosy showing Th2 immune response and high bacterial load,

which is therefore classified toward the lepromatous pole (BB,

BL, and LL) of the disease. Most T2Rs occur during MDT

[10], and the majority of patients usually experience several

acute or chronic episodes over the years. Type 2 reaction pres-

ents as disseminated painful erythematous tender nodules with

or without neuritis, usually followed by systemic symptoms

such as fever and malaise. Type 2 reaction is characterized by

increased levels of tumor necrosis factor a and immunecomplexassociated vasculitis, panniculitis, and uveitis. Treat-

ment for T1Rs is based on corticosteroids, and T2R therapy

includes corticosteroids and/or thalidomide [5].

Genomic sequence studies of M. leprae strains of worldwide

distribution have shown very low genetic diversity, suggesting

that differences in susceptibility to disease and to clinical man-

ifestations, including reactional episodes, are influenced by the

host defense genes [11]. Only recently genetic epidemiology

studies have identified predictive susceptibility factors for

leprosy reactions. Most of these studies focused on variants on

genes for Toll-like receptors 1 and 2 (TLR1, TLR2), important

mediators of mycobacterium recognition during innate im-

mune response. Alleles of both the single-nucleotide poly-

morphism (SNP) rs3804099 and a microsatellite intragenic to

TLR2 were associated with T1R in an Ethiopian population

[12]. Nonsynonymous SNP rs5743618 (I602S) of TLR1 was

found to be associated with protection against T1R in a Nepali

population [13]. The same variation was subsequently associated

with leprosy per se in Indian and Turkish populations [14].

Another nonsynonymous polymorphism of TLR1 (N248S) was

associated with leprosy per se and T2R, with the S and the

N alleles more frequent among all patients with leprosy and

patients with T2R, respectively [15]. Finally, the first genome-

wide association study in leprosy revealed 4 SNPs intragenic

to the NOD2 gene associated with leprosy [16]. A subsequent

study showed variants of the same gene associated with lep-

rosy per se, T1R, and T2R [17]. However, associated variants

were not the same in the 2 studies.

A recent analysis of 27 plasma factors, including cytokines,

chemokines, and growth factors, by our group revealed in-

terleukin 6 (IL-6) as the only biomarker of both T1R and T2R

when compared with leprosy-affected individuals without re-

action [18]. Previous studies have found higher IL-6 levels as-

sociated with LL leprosy, which is associated with occurrence

of T2R [1921]. A constitutive gene expression analysis using

nonstimulated peripheral blood mononuclear cells from pa-

tients with reactional or nonreactional leprosy revealed that

IL-6 was expressed in most of the patients with T1R or T2R.

Among individuals with nonreactional leprosy, IL-6 messenger

RNA was detected only in patients with TT or BT leprosy.

When skin biopsy specimens were analyzed, expression of IL-6

was detected in most lesions of both T2R and TR1 patients [19].

Nevertheless, it is known that messenger RNA expression and

the actual level of the secreted protein may be different in vivo.

The production of IL-6 by peripheral blood mononuclear cells

in response to anti-CD3 T-cell polyclonal stimulant was ob-

served in a significantly higher proportion of patients with

LL leprosy, compared with TT leprosy [20]. In a comparative

study, lepromatous patients had the highest serum levels of

IL-6, and patients with T2R had higher levels of IL-6 than those

with nonreactional leprosy [21]. As a continuation of our pre-

vious study [18], here we present the results of a follow-up,

prospective, nested, case-control study of association between

leprosy reactions and tag polymorphisms of the IL-6 gene in

an expanded population sample of affected individuals re-

cruited at the same Brazilian reference center for leprosy

diagnosis and treatment.

MATERIALS AND METHODS

Population SampleA cohort of patients with leprosy was recruited at the time of

leprosy diagnosis (newly detected cases) at a public health

reference center (Centro de Referencia em Diagnostico e

Terapeutica) located in Goiania city, central western Brazil,

between February 2006 and March 2008. Leprosy diagnosis/

classification was defined after detailed dermatoneurological

examination by a clinician with expertise in leprosy diagnosis,

bacilloscopy, and histopathology of skin lesion biopsy speci-

mens, according to Ridley-Jopling criteria [2]. Case definitions

of leprosy, T1R, and T2R have been described elsewhere [21].

Inclusion criteria were newly detected patients, without history

of previous leprosy treatment, presenting or not presenting lep-

rosy reactions (T1R or T2R). Patients with leprosy were closely

followed up for reactional episodes during MDT (6 months for

paucibacillary patients and 12 months for multibacillary pa-

tients). On completion of MDT, patients were strongly ad-

vised to return to the reference center if any sign or symptom

of reaction occurred. Overall, patients have been followed up

for $2 years since diagnosis; leprosy reactions were observed

at diagnosis, during MDT, and after MDT. For patients who

came for diagnosis during a reactional episode, blood was col-

lected before the start of MDT and antileprosy reaction therapy.

For patients with leprosy who developed reactions during

MDT, blood was collected during MDT. For patients who

had a reactional episode after completion of MDT, by the

time blood was collected patients were taking neither MDT

nor any other specific drug therapy for leprosy reaction.

Exclusion criteria for the genetic analysis were uncertain

classification of leprosy or type of leprosy reaction, clinical

classification inconsistent with the type of reaction, and clinical

signs and symptoms compatible with pure neuritis. Two nested

1418 d JID 2012:205 (1 May) d Sousa et al



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case-control studies were built from this cohort: the first included

patients with leprosy at their first episode of T1R, and the second

included patients with leprosy at their first episode of T2R.

Controls were patients with newly detected leprosy who did

not have reaction at the time of initial diagnosis or during

follow-up. Patients with T1R were compared with controls

presenting with polar TT or intermediary BT, BB, or BL leprosy.

Patients with T2R were compared with controls with polar LL

or BL and BB disease. All patients were treated for leprosy ac-

cording to World Health Organization MDT guidelines and for

leprosy reaction with the appropriate therapy. Demographic

and clinical variables were compared using v2 and t tests;differences were considered significant at an a error of ,.05(P value). This study was approved by the Brazilian National

Committee for Ethics in Research (CONEP) (protocol 650/

2006/12638). All patients signed an informed consent; for

patients ,18 years old, the informed consent was signed by one

of the parents or the legal guardian.

Marker SelectionTag SNP markers capturing the entire information of the IL-6

locus were selected according to the information available at

the International HapMap Project website (release 24/phase

2_Nov08). All selectedmarkers presented aminor allele frequency

of 0.2 or higher in the CEU HapMap population (northern or

western European ancestry). Two markers were considered in

linkage disequilibrium (LD) when presenting a pairwise r2. 0.8.

Following these criteria, tag SNPs rs2069832, rs2069840, and

rs2069845 were selected for genotyping.

DNA Extraction and GenotypingGenomic DNA was obtained from peripheral blood by classic

salting-out protocol [22] and was then purified and quantified.

Tag SNPs rs2069832, rs2069840, and rs2069845 were geno-

typed with a polarized fluorescence, Taqman probebased

method [23], using the ABI 7500 platform. Genotypes were

automatically assigned using the appropriate software with

subsequent manual checking of accuracy. Functional SNP

rs1800795 was genotyped by restriction fragment length poly-

morphism analysis, as described elsewhere [24].

Plasma IL-6 LevelsPlasma levels of IL-6 were measured for all patients with

leprosy for whom a biological sample was available. The

IL-6 levels were determined by enzyme-linked immunosor-

bent assay (eBioscience Human IL-6 kit) according to the

manufacturers instructions, using undiluted samples. An

exploratory stem-and-leaf test was carried out to identify

outliers.

Statistical AnalysisHardy-Weinberg equilibrium estimates were carried out using

Haploview 4.2 software [25]. Genotyping frequencies for cases

and controls were compared by univariate analysis under both

dominant and additive model using v2 test or Students t testswhen appropriate. Adjustment for sex, clinical form, and age

was performed using stepwise multivariate logistic regression

analysis. The P value threshold for rejection of the null hy-

pothesis was fixed at .05; for comparisons that reached statistical

significance, odds ratios were estimated with the appropriate

test. The MannWhitney test was used to compare IL-6 plasma

levels across controls and patients with leprosy reactions as

well as across genotypes under a dominant model. Analyses

were performed with the statistical software SPSS 13.0.

RESULTS

A total of 429 leprosy-affected individuals were recruited for

the study. Based on exclusion criteria, 20 individuals were

removed from the analysis: 10 with pure neuritis and 10 for

inconsistencies or uncertain diagnosis of either leprosy and/or

type of reaction. During the 2 years of follow-up since di-

agnosis, 37.7% of patients with leprosy (154 of 409) de-

veloped T1R, 9.5% (39 of 409) developed T2R, and the

remaining 52.8% (216 of 409) did not present any sign of

reaction. Regarding T1R, 8.5% of patients (13 of 154) came

for diagnosis during the first T1R episode, 56.5% (87 of 154)

presented the first TR1 episode during MDT, and the remaining

36% (54 of 154) developed T1R after completion of MDT, at

a median of 3 months (range, 114) after MDT. Among patients

with T2R, 10.2% of patients (4 of 39) came for diagnosis during

the first reactional episode, 63.8% (25 of 39) presented T2R

during MDT, and 26% (10 of 39) presented T2R after MDT, at

a median of 4 months (range, 113) after MDT.

For the genetic analysis, the T1R subgroup was compared

with 188 controls with nonreactional leprosy presenting with

TT, BT, BB, or BL leprosy; the T2R subgroup was compared

with 70 controls with nonreactional leprosy presenting BB,

BL, or LL leprosy. Comparison between patients with T1R

and controls revealed statistically significant differences in

the distribution of the leprosy classification (P 5 8.1 3 1025),

sex (P 5 .04) and age (P 5 1.3 3 1025). Type 1 reaction was

more frequent among individuals with BT leprosy (51.3% of

patients) and male patients (61.7%); the mean age for patients

with T1R was higher (51.0 6 15.2 years) than for controls

(41.7 6 16.6 years) (P 5 1.3 3 1025). For the T2R subgroup,

the only difference observed between patients and controls

was related to the leprosy classification (P 5 .005): the LL

classification was more frequent among patients with T2R

(71.8%) than among controls (40%). Description and analysis

of the Ridley-Jopling classification and epidemiological data

from T1R and T2R controls are summarized in Table 1.

To replicate the association between IL-6 and occurrence of

leprosy reactions observed previously in a smaller subsample

of the studied population [18], we compared plasma levels of

IL-6 among 16 reaction-free, leprosy-affected control




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individuals, 33 patients with T2R, and 54 patients of T1R from

our expanded data set (Figure 1). Results confirmed increased

levels of IL-6 among patients with both T1R (P 5 .009) and

T2R (P 5 2.4 3 1028) when compared with reaction-free

leprosy controls. Interestingly, the magnitude of the effect was

much more pronounced for T2R than for T1R (P5 1.03 1026).

All genetic markers tested were in Hardy-Weinberg equilib-

rium. Genetic analyses failed to detect association between

IL-6 variants and the occurrence of T1R. No association was

observed even after the removal of the 22 controls with TT

leprosy from this subpopulation (data not shown), because

these patients present a relatively stable immune response and

are less susceptible to developing T1R. Positive evidence for

association between all 3 tag SNPs and occurrence of T2R

(Table 2) was observed for both dominant and additive

models after correction for leprosy clinical form. Linkage

disequilibrium analysis confirmed independence of all 3 SNPs

(Figure 2).

A comprehensive search on the HapMap database (build

24/phase 2_Nov08) revealed that SNP rs2069832 was tagging

a bin that contained variant rs1800795, a known, functional

polymorphism initially localized at a negative regulatory domain

of the promoter region of IL-6 [26]. Therefore, SNP rs1800795

was genotyped exclusively in the T2R subpopulation. Analysis

confirmed strong LD between rs1800795 and rs2069832 in our

population sample (r25 0.69) (Figure 1). Moreover, association

between rs1800795 functional variant of IL-6 and occurrence

of T2R was also observed. Odds ratios were estimated at 4.0

(95% confidence interval [CI], 1.649.76) and 3.7 (95%

CI, 1.479.34) for risk alleles A and C of markers rs2069832

and rs1800795, respectively. Allele G of marker rs2069840

showed very weak LD with both rs2069832 and rs1800795

SNPs and was associated with protection against T2R (odds

ratio, 0.27; 95% CI, .22.91).

Multivariate analysis including all 4 SNPs confirmed in-

dependent association of 2 bins: the first captured by tag SNP

rs2069832 and including markers rs2069845 and rs1800795

(P 5 .01) and the second an rs2069840 singleton, the latter

with borderline significance (P 5 .06). Haplotypic analysis

also revealed a statistically significant association between

T2R and haplotypes composed by the risk alleles of rs2069840

and both functional SNP rs1800795 and tag SNP rs2069832

(P 5 .02 and .04, respectively) (Supplementary Table 1).

Table 1. Ridley-Jopling and Epidemiological Data of Patients With Leprosy Type 1 Reaction (T1R), Patients With Leprosy Type 2 Reaction(TR2), and Controls

T1R (n 5 154) Controls (n 5 188) P a T2R (n 5 39) Controls (n 5 70) P a

Ridley-Jopling classification 2.46 3 1028 .005

TT . 22 (11.7) . .

BT 79 (51.3) 124 (66.0) . .

BB 29 (18.8) 16 (8.5) 3 (7.7) 16 (22.8)

BL 46 (29.9) 26 (13.8) 8 (20.5) 26 (37.2)

LL . . 28 (71.8) 28 (40.0)

Sex .01 .60

Male 95 (61.7) 91 (48.4) 27 (69.2) 45 (64.3)

Female 59 (38.3) 97 (51.6) 12 (30.8) 25 (35.7)

Age, years 3.8 3 1027 .07

Mean 6 SD 51.0 6 15.2 41.7 6 16.6 40.0 6 16.6 45.9 6 16.9

Range 1088 787 1178 979

Unless otherwise specified, data represent No. (%) of patients or controls.

Abbreviations: TT, tuberculoid leprosy; BT, borderline tuberculoid; BB, borderline borderline; BL, borderline leprosy; LL, lepromatous leprosy; SD, standard

deviation.a P values were determined with v2 test for Ridley-Jopling classification and sex and with Students t test for age.

Figure 1. Plasma levels (pg/mL) of interleukin 6 (IL-6) among 16controls with reaction-free leprosy, 54 patients with type 1 reaction (T1R),and 33 patients with type 2 reaction (T2R) from the genetic study data set.P represents nonparametric MannWhitney P values.




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Investigation of association between genotypes and plasma

levels of IL-6 was performed using the entire subsample of

39 patients with T2R (Figure 3). Protective allele G of marker

rs2069840 was associated with lower IL-6 plasma concen-

trations under a dominant model (P 5 .04). Higher plasma

levels of IL-6 were observed among carriers of susceptibility

alleles A and C of markers rs2069832 and rs1800795, although

the differences did not reach statistical significance. Five in-

dividuals presented a plasma IL-6 concentration $ 56 pg/mL

(considered outliers) and were removed from the analysis after

stem-and-leaf filtering.

DISCUSSION

This study presents combined genetic and serological evidence

that implicates IL-6 gene variants as risk factors for susceptibility

to the occurrence of leprosy T2R. The IL-6 gene was selected for

genetic analysis based on results of a previous multiplex cytokine

study from our group showing that, among 27 plasma factors

tested, only IL-6 was identified as a potential biomarker of both

T1R and T2R [18]. First, we replicated the original finding for

both T1R and T2R in an expanded population sample of pa-

tients recruited from the same highly endemic region following

a prospective design. However, the effect was less notable for

the T1R group (Figure 1). Then, our strategy of comprehensive

physical mapping of the IL-6 gene using tag SNPs followed by

a genotype-phenotype correlation study led to an unbiased

identification of a functional IL-6 variant associated with T2R

in the expanded population sample. No association between IL-

6 polymorphisms and leprosy T1R was observed. This is an

intriguing finding that deserves further consideration. Occur-

rence of both types of leprosy reaction is clearly associated with

increased production of pleiotropic inflammatory cytokine

IL-6; however, in T1R, higher levels of IL-6 take place in

a context of an already existing Th1 type of immune response.

This increase of IL-6 in T1R could be due to nongenetic an-

d/or genetic determinants located in different genes of the

immune pathway. In contrast, a more exuberant increase of

plasma IL-6 among individuals with T2R seems to be de-

pendent on genetic determinants playing a central role

Table 2. Association Between Interleukin 6 (IL-6) Genetic Variants and Occurrence of Leprosy Type 1 Reaction (T1R) and Type 2Reaction (T2R)

Variant T1R Controls P a P b T2R Controls P a P b Odds Ratio (95% CI)c

IL-6 rs1800795

CC . . . . 1 (2.6) 3 (4.5) .02 .03 2.41 (1.095.33)

CG . . 22 (57.9) 20 (29.8)

GG . . 15 (39.5) 44 (65.7)

CC 1 CG . . . . 23 (60.5) 23 (34.3) .009 .005 3.71 (1.479.34)

GG . . 15 (39.5) 44 (65.7)

IL-6 rs2069832

AA 7 (4.7) 5 (2.7) .33 .23 4 (10.5) 3 (4.4) .005 .007 2.71 (1.325.57)

AG 39 (26.2) 66 (36.1) 20 (52.6) 19 (27.9)

GG 103 (69.1) 112 (61.2) 14 (36.9) 46 (67.7)

AA 1 AG 46 (30.9) 71 (38.8) .13 .11 24 (63.1) 22 (32.3) .002 .002 4.00 (1.649.76)

GG 103 (69.1) 112 (61.2) 14 (36.9) 46 (67.7)

IL-6 rs2069840

GG 16 (10.7) 17 (9.2) .50 .50 1 (2.6) 8 (12.1) .03 .03 0.44 (.22.91)

CG 73 (48.7) 86 (46.7) 13 (33.3) 28 (42.4)

CC 61 (40.6) 81 (44.1) 25 (64.1) 30 (45.5)

GG 1 CG 89 (59.4) 103 (55.9) .54 .51 14 (35.9) 36 (54.5) .06 .04 0.39 (.16.96)

CC 61 (40.6) 81 (44.1) 25 (64.1) 30 (45.5)

IL-6 rs2069845

GG 13 (8.6) 17 (9.3) .45 .29 8 (20.5) 7 (10.4) .02 .04 1.92 (1.023.63)

AG 63 (41.7) 83 (45.6) 22 (56.4) 30 (44.8)

AA 75 (49.7) 82 (45.1) 9 (20.1) 30 (44.8)

GG 1 AG 76 (50.3) 100 (54.9) .40 .30 30 (79.9) 37 (55.2) .03 .045 2.59 (1.026.56)

AA 75 (49.7) 82 (45.1) 9 (20.1) 30 (44.8)

Unless otherwise specified, data represent No. (%) of patients or controls.

Abbreviation: CI, confidence interval.a P values for v2 test.b P values for multivariate logistic regression analysis.c Odds ratios from multivariate logistic regression analysis.




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because T2R occurs in a context of Th2 immune response

background with only transient Th1 activation. This hypothesis

is strongly reinforced by the results of both genetic association

and genotype-phenotype correlation shown here. Altogether,

our results argue in favor of different physiopathological

mechanisms for T1R and T2R, in which genetic and non-

genetic variants may have different effects on the phenotypes.

The small sample of patients with leprosy T2R is an ac-

knowledged limitation of our study. Despite the large number

of leprosy-affected individuals enrolled for follow-up, the

number who developed T2R was small, as expected, reducing

the power of the association analysis. Another limitation is the

lack of replication of the genetic findings in an independent

population sample. Nevertheless, these limitations are partially

minimized by the fact that genetic association between at least

1 SNP bin, represented by tag SNP rs2069840, and occurrence

of T2R was also observed in an independent experiment using

plasma levels of IL-6 as a phenotype. The second associated bin,

containing known functional SNP rs1800795, also correlated with

different IL-6 plasma levels, but this association did not reach

statistical significance, possibly because of the small sample size.

In humans, the IL-6 gene, located at 7p21 chromosome,

produces a secreted 26-kDa glycoprotein arranged in 4 chains.

There are several possible mechanisms by which higher IL-6

levels might affect the pathogenesis of T2R. Although IL-6 was

first recognized by its property to induce antibody production,

it is considered a key player in inflammation and in acute-

phase reaction, which is one of the earliest responses to insults.

Interleukin 6 can be produced by a broad spectrum of cell

types, including T and B cells, macrophages, activated

monocytes, mast cells, neutrophils, and eosinophils, and can

exert its effects on virtually any cell. Interleukin 6 is known to be

deregulated in many chronic autoimmune and inflammatory

diseases, including rheumatoid arthritis, Crohns disease, juve-

nile arthritis, and multiple myeloma [26]. Interleukin 6 also

participates in the adaptive immune response affecting T-cell

differentiation into regulatory T cells or Th1 and Th2 and

particularly T-helper 17 differentiation in humans. Although

speculative, it seems reasonable to assume that higher levels of

IL-6 could contribute to the development of leprosy T2R,

mainly owing to the potent proinflammatory role of IL-6

and its capacity to stimulate antibody production. Together

with extensive systemic inflammatory manifestations, im-

mune complexassociated vasculitis is one of the hallmarks

of leprosy T2R.

The results presented in this report are, to our knowledge,

the first implicating IL-6 polymorphisms in leprosy T2R.

Previous studies implicate IL-6 genetic variants with differ-

ent diseases. In particular, SNP rs1800795 is a well-known

functional IL-6 polymorphism located at position 174 of

the promoter region of the gene, inside a negative regulatory

domain. Alleles of rs1800795 have been described as risk factors

for Graves disease [27], systemic-onset juvenile chronic arthritis

[26], periodontitis [24, 28, 29], hepatitis C [30], and both types 1

and 2 diabetes mellitus [31, 32], with inconclusive results for type

2 diabetes mellitus [33]. As for tropical infectious diseases,

the same allele C of rs1800795, identified here as a risk factor for

T2R, has been associated with mucosal leishmaniasis in a Brazilian

population [34]. Other studies failed to detect associations be-

tween IL-6markers, including rs1800795, and Chagas disease and

human immunodeficiency virus infection [35, 36].

Individuals homozygous for allele C of rs1800795 were

shown to have a 6-fold higher risk of developing Crohns

disease, a chronic inflammatory bowel disease that can also

have erythema nodosum as a systemic complication [37].

Interestingly, this is not the first time that alleles of a gene have

been associated with both Crohns disease and leprosy pheno-

types. Several genetic epidemiology studies have independently

detected variants of NOD2, TNFSF15, and IL12B as risk fac-

tors for both Crohns disease and leprosy [16, 3840], leading

authors to suggest an exciting hypothesis of a mycobacterial

origin for Crohns disease [41]. In addition, NOD2 poly-

morphisms have been also associated with both types of

leprosy reactions [17].

To date, no previous association of rs2069840 has been

detected with any chronic inflammatory autoimmune or

infectious disease. Because this SNP is not in LD with func-

tional rs1800795 and seems to be independently associated

with occurrence of T2R, these observations suggest the exis-

tence of additional IL-6 functional variants other than the

rs1800795 LD bin that play a role in T2R susceptibility. In

fact, marker rs2069840 seems to be of particular importance

Figure 2. Relative position and linkage disequilibrium (LD) pattern ofmarkers for the interleukin 6 gene (IL-6). Values inside boxes represent LDmeasured using the r2 parameter.




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because it is associated with both T2R and plasma levels of

IL-6. Interestingly, haplotypes composed of risk alleles of

rs2069840 and both functional SNP rs1800795 and rs2069832

were also associated with T2R, even in low-power analyses due

to low allele frequencies.

The essential role of IL-6 in leprosy T2R immunopathology

needs to be explored further. The use of IL-6 as a biomarker in

several diseases has been widely discussed, based on the multiple

effects of this cytokine on the control of innate and adaptative

immunity [42]. Here we propose genetic variants of IL-6 as

possible biomarkers for T2R in leprosy. The results presented

here add up to a solid body of evidence implicating IL-6 in the

pathogenesis of leprosy T2R and point to this cytokine as

a possible valuable predictive marker for this aggressive leprosy

phenotype.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases

online (http://www.oxfordjournals.org/our_journals/jid/). Supplementary

materials consist of data provided by the author that are published to benefit

the reader. The posted materials are not copyedited. The contents of all

supplementary data are the sole responsibility of the authors. Questions or

messages regarding errors should be addressed to the author.

Notes

Acknowledgments. We are grateful to the patients and staff of the

Reference Center for Diagnosis and Treatment, Goiania city, for their

cooperation and assistance during this study.

Financial support. This work was supported by the Brazilian National

Council of Technological and Scientific Development/CNPq (grant

401276/2005-8). M. M. A. S. is supported by CNPq (fellowship 304869/

2008-2) and is a member of the IDEAL Consortium (Initiative for Di-

agnostic and Epidemiological Assays for Leprosy). M. T. M. is supported

Figure 3. Plasma levels (pg/mL) of interleukin 6 (IL-6) according to IL-6 genotypes. Subsample was composed of 39 patients with type 2 reactiondistributed into different genotype classes. P represents the MannWhitney P value for the IL-6 plasma levels across genotypes under a dominant model.Circles represent individuals with IL-6 plasma levels above the 95% confidence interval.




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by Ministerio de Ciencia e Tecnologia/CNPq/Ministerio da Saude SCTIE-

DECIT 25/2006 (Estudo de Doencxas Negligenciadas) (grant 410470/2006-6), the Order of Malta Grants for Leprosy Research 2008, and the

INSERM-CNPq joint program, project 490844/2008-1 (ASCIN/CNPq

61/2008 Convenios Bilaterais Europa). L. H. F. S. was supported by a

scholarship from the Coordination of Improvement of Higher Education

Personnel/CAPES.

Potential conflicts of interest. All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential

Conflicts of Interest. Conflicts that the editors consider relevant to the

content of the manuscript have been disclosed.

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