Influence of HLA-DRB1 alleles on Th1 and Th2 cytokine response to Mycobacterium tuberculosis antigens in pulmonary tuberculosis

ARTICLE IN PRESS Tuberculosis (2007) 87, 544–550

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Influence of HLA-DRB1 alleles on Th1 and Th2 cytokine response to Mycobacterium tuberculosis antigens in pulmonary tuberculosis P. Selvaraj, D. Nisha Rajeswari, M.S. Jawahar, P.R. Narayanan Tuberculosis Research Centre, Indian Council of Medical Research, Mayor V.R. Ramanathan Road, Chetput, Chennai 600 031, India Received 8 March 2007; received in revised form 2 August 2007; accepted 2 August 2007

KEYWORDS HLA-DRB1 alleles; Cytokines; Pulmonary tuberculosis

Summary The influence of human leukocyte antigens (HLA) on the immune response is well established. We investigated the regulatory role of HLA-DRB1 alleles on cytokine response to live M. tuberculosis and its culture filtrate antigen (CFA) in normal healthy subjects (NHS) and pulmonary tuberculosis (PTB) patients. Th1 (IFN-g and IL-12p40), Th2 (IL-4 and IL-5), pro-inflammatory (IL-6 and IL-8) and anti-inflammatory (TGF-b and IL-10) cytokines were measured by ELISA in 72-h-old peripheral blood mononuclear cell culture supernatants from 58 NHS and 48 PTB patients. HLA-DRB1 genotyping was carried out by polymerase chain reaction and dot-blot hybridization with biotinylated sequence-specific oligonucleotide probes and detection by chemiluminescence. In response to live M. tuberculosis and CFA, significantly increased levels of IL-6, IL-8 and TGF-b and decreased IFN-g, IL-12p40 and IL-10 were seen in PTB patients compared to NHS. We observed a significantly increased IFN-g response in HLA-DRB1*03-positive NHS (p ¼ 0.03) and decreased IFN-g response in HLA-DRB1*15-positive patients (p ¼ 0.04) than respective allele-negative individuals. An increased level of IL-12p40 in DRB1*10 (p ¼ 0.02) and IL-10 in DRB1*12- (p ¼ 0.03) positive NHS and an increased level of IL-6 in DRB1*04- (p ¼ 0.02) positive patients were observed. The study suggests that HLA-DRB1 alleles differentially modulate the various cytokine responses to M. tuberculosis antigens, which may influence the cellular and humoral immune responses to M. tuberculosis infection in a susceptible host. & 2007 Elsevier Ltd. All rights reserved.

Introduction Corresponding author. Tel.: +91 44 2836 9761;

fax: +91 44 2836 2528. E-mail address: [email protected] (P. Selvaraj). 1472-9792/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2007.08.001

A perplexing yet unsolved feature of tuberculosis (TB) is that less than 10% of infected individuals develop the disease. Whether a person develops TB depends on host pathogen

ARTICLE IN PRESS HLA-DR and cytokine profile in tuberculosis interactions dictated partly by the host genetic factors. Resistance to M. tuberculosis infection is known to be conferred by T cell-mediated immune mechanisms. Among the T cells, CD4+ T helper cells play a crucial role in protective immunity to TB.1 Upon antigenic stimulation, CD4+ Th cells differentiate into Th1 cells, which secrete cytokines that are involved in cell-mediated immune response (IFN-g and IL-12) or Th2 cells that secrete mediators of humoral immunity (IL-4 and IL-5).2 Other pro-inflammatory (IL-6 and IL-8) and antiinflammatory cytokines (TGF-b and IL-10) also regulate immune response to TB. A pathway of T cell activation, initiated by TGF-b and IL-6 resulting in responsiveness to IL23, results in antigen-specific T cells that produce IL-17 (Th17).3 Th17 cells have a crucial role in induction of autoimmune tissue injury and are regulated by IFN-g.4 IFN-g, the signature Th1 cytokine, activates infected macrophages to eliminate intracellular pathogens. IL-12, which comprises of p35 and p40 subunits, directs the development of Th1 cells, while IL-4, the principal Th2 cytokine, suppresses the Th1 response.5 IL-10 secreted by alternatively activated macrophages and T cells is known to downregulate IL-12 production.6 IL-8 (CXCL8) is a chemokine secreted by macrophages and T cells that attracts neutrophils and T cells.7 TGF-b is implicated in suppression of T cell and antibacterial immune responses in TB.8 IL-6 secreted by T cells and macrophages regulates various cell types.3,9 CD4+ T cells are activated by the recognition of pathogenderived peptides in the context of HLA class II molecules presented by antigen presenting cells. The highly polymorphic nature of human leukocyte antigens (HLA) allows it to bind a repertoire of peptides that are specific for each HLA molecule, thereby influencing T cell polarization and hence the profile of cytokines secreted.10 The major histocompatibility complex linked control of CD4+ T cell activation with distinct cytokine profile has been established in murine models.11 In humans, significant difference in cytokine secretion profiles has been observed in peripheral blood mononuclear cells (PBMCs) from HLA-B8, -DR3positive individuals.12 HLA-DR2 has been shown to be associated with susceptibility to pulmonary tuberculosis (PTB) among Indian populations.13–15 The immune mechanisms behind HLA-DR associated susceptibility to TB remains unclear. Our earlier studies have shown that HLA-DR antigens can influence humoral and cell-mediated immune responses to TB.16 In the present study, we investigated the influence of HLA-DRB1 alleles on live M. tuberculosis and its culture filtrate antigen (CFA) induced cytokine response in PTB patients and healthy controls.

545 immunodeficiency virus (HIV) negative and none was known to present any immunosuppressive condition. Fifty-eight endemic normal healthy subjects (NHS) comprising 38 males (mean age7S.D. ¼ 31.277.7 yr) and 20 females (mean age7S.D. ¼ 26.874 yr) were also studied. The study was approved by the institutional ethical committee. Patients and controls were from the same ethnic group of the south Indian population from the state of Tamil Nadu.

Cell preparation and in vitro culture PBMCs were isolated by density gradient centrifugation on Ficoll-Hypaque. PBMCs were suspended at a concentration of 2  106 cells/ml in RPMI 1640 (Sigma Aldrich, St. Louis, MO, USA) supplemented with 2 mM glutamine, 0.1 mM sodium bicarbonate and 2% autologous serum and plated in 48 well plate (Costar, Cambridge, MA, USA). Live M. tuberculosis H37Rv (multiplicity of infection 1 macrophage: 10 bacilli) or M. tuberculosis CFA 10 mg/ml, prepared as described earlier,17 was added to respective wells. After 72 h of culture at 37 1C and 5% CO2, supernatants were harvested and frozen at 80 1C. Seventy-two hours has been shown to be the optimal time for cytokine secretion.18–20

Measurement of cytokine levels by ELISA Cytokine levels of IFN-g, IL-12p40, IL-4, IL-5, IL-6, IL-8, IL-10 and TGF-b in the culture supernatants were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA). The detection limits for these assays were 15–1000 pg/ml for IFN-g and IL-6; 30–2000 pg/ml for IL-10, IL-8 and TGF-b; 62–4000 pg/ml for IL-12p40; 31.2–2000 pg/ml for IL-4 and 23.4–1500 pg/ml for IL-5.

Genotyping of HLA-DRB1 alleles Genomic DNA was isolated by the salting-out technique from the granulocytes.21 HLA-DRB1 typing was done by polymerase chain reaction and dot-blot hybridization with biotinylated sequence specific oligonucleotide probes, followed by detection using chemiluminescence method.22

Statistical analysis

Patients and methods

Results were analysed using Student’s ‘t’ test (unpaired, two tailed) for comparison of cytokine levels between DRB1 allele-positive and DRB1 allele-negative group. Data were shown as mean7SEM (standard error of mean) and were considered statistically significant when the p value was less than or ¼ 0.05.

Study subjects

Results

Forty-eight patients with PTB comprising 31 males (mean age7S.D. ¼ 36.5710.9 yr) and 17 females (mean age7 S.D. ¼ 32.8710.2 yr), from Tuberculosis Research Centre, Chennai, were included in the study before anti-TB treatment was started. Diagnosis of TB was made by chest radiography, Ziehl–Neilsen staining of sputum smears and was confirmed by sputum culture. All patients were human

Cytokine profile in patients and controls An increase in IL-6, IL-8, TGF-b level and a decrease in IL-10, IFN-g, IL-12p40 level in response to live M. tuberculosis and CFA of M. tuberculosis were observed in PTB patients when compared to NHS. In PTB patients and NHS, a significant increase in the cytokine level of IL-6, IL-8, IL-10, IFN-g,

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P. Selvaraj et al. DRB1*15 allele was observed among patients compared to controls; however, due to a small sample size, the difference was not significant. In NHS and PTB patients, there was no significant difference in the spontaneous IL-6, IL-8, TGF-b, IFN-g, IL-12p40, IL-4 and IL-5 response among various HLA-DRB1 allele-positive and their respective DRB1 allele-negative groups (data not shown). PBMCs of DRB1* 15-positive PTB patients showed an increased spontaneous IL-10 response (p ¼ 0.03) compared to non-DRB1*15 patients (data not shown). We observed no significant difference in CFA and live M. tuberculosis induced IL-4, IL-5, TGF-b and IL-8 response between various HLA DRB1 allele-positive and respective DRB1 allele-negative groups in both patients and NHS (data not shown). DRB1*03-positive NHS (n ¼ 10) showed a significantly increased CFA (p ¼ 0.01) and live M. tuberculosis (p ¼ 0.03) induced IFN-g level compared to DRB1*03-negative NHS (n ¼ 48) (Fig. 3A). In PTB patients, there was a decreased IFN-g response to CFA (p ¼ 0.04) and live M. tuberculosis (p ¼ 0.04) in DRB1*15-positive patients (n ¼ 18) compared to non-DRB1*15 patients (n ¼ 30) (Fig. 3B). Differences in the cytokine responses between HLA DRB1*16-positive and -negative individuals cannot be analysed due to a low number of DRB1*16-positive individuals. An increased IL-12p40 response to live M. tuberculosis (p ¼ 0.02) in DRB1*10-positive NHS (n ¼ 17) compared to DRB1*10-negative NHS (n ¼ 41) was observed and no difference was seen in PTB patients (Fig. 3C). There was no association between HLA-DRB1 alleles and IL-5 response to M. tuberculosis in patients and controls (data not shown).

IL-12p40, IL-5 (po0.05) was observed in CFA and live M. tuberculosis stimulated cultures compared to unstimulated cultures (controls). No difference was observed for IL-4 and TGF-b between stimulated and unstimulated cultures. An increased spontaneous (p ¼ 0.02), CFA (p ¼ 0.02) and live M. tuberculosis (p ¼ 0.02) induced IL-6 response was observed in patients when compared to NHS (Fig. 1A). PTB patients showed an increased IL-8 response to CFA (p ¼ 0.004) and live M. tuberculosis (p ¼ 0.006) (Fig. 1B) and a decreased spontaneous, CFA and live M. tuberculosis induced IL-10 response (p ¼ 0.02, p ¼ 0.0005, p ¼ 0.004) compared to healthy subjects (Fig. 1C). An increased TGF-b response to CFA (p ¼ 0.01) and live M. tuberculosis (p ¼ 0.002) was seen in patients when compared to NHS (Fig. 1D). There was no difference in the spontaneous response of IL-8 and TGF-b between patients and controls. Decreased spontaneous response of IFN-g and IL-12p40 (p ¼ 0.01, Po0.0001) was observed in patients, similar to a decreased CFA (Po0.0001, p ¼ 0.002) and live M. tuberculosis (Po0.0001, p ¼ 0.002) induced response in patients compared to NHS (Fig. 2A and B). No difference in the spontaneous, CFA and live M. tuberculosis-induced IL-4 and IL-5 response was observed between patients and controls (Fig. 2C and D).

Association between HLA DRB1 alleles and cytokine response The frequencies of the HLA DRB1 alleles in the study population are given in Table 1. An increased frequency of

NHS(n=58) 200

P=0.02

∗∗

180

∗∗∗

∗∗

140

∗∗∗

120 100 80 60 40 20

P=0.02

∗ ∗

400

∗

P=0.006

∗∗ ∗∗

300 200

0 CONTROL

2000

CFA

MTB

CONTROL

∗∗

P=0.02

∗∗∗

∗

∗

TGF-BETA (pg/ml)

∗∗∗

1000

MTB

P=0.01 P=0.004

1500

CFA

2000

P=0.0005

∗∗ IL-10 (pg/ml)

∗

100

0

500

P=0.004

500 IL-8 (ng/ml)

IL-6 (ng/ml)

160

PTB(n=48)

600

P=0.02

1500

P=0.002

∗∗

∗

1000

∗∗

500

∗ 0

0 CONTROL

CFA

MTB

CONTROL

CFA

MTB

Fig. 1 The pro-inflammatory and anti-inflammatory cytokine profile of spontaneous, CFA, M. tuberculosis (MTB) stimulated PBMC in normal healthy subjects (NHS) (n ¼ 58) and pulmonary tuberculosis patients (PTB) (n ¼ 48). (A) IL-6. (B) IL-8. (C) IL-10. (D) TGF-b.

ARTICLE IN PRESS HLA-DR and cytokine profile in tuberculosis

547 NHS (n=58) PTB (n=48)

P<0.0001 ∗∗

IFN-GAMMA (ng/ml)

20 15

10

∗∗

5

∗∗∗

∗∗∗

2500

P=0.002

∗

2000 1500

P<0.0001

∗

∗∗∗ ∗

1000 500

∗

CONTROL

P=0.002

3000

P=0.01 ∗

0

3500

P<0.0001 ∗∗∗

IL-12P40 (pg/ml)

25

∗

0 CFA

MTB

CONTROL

CFA

MTB

CONTROL

CFA

MTB

140

25

120

20 IL-5 (pg/ml)

IL-4 (pg/ml)

100 15 10

80 60 40

5

20

0

0 CONTROL

CFA

MTB

Fig. 2 The Th1 and Th2 cytokine profile of spontaneous, CFA, M. tuberculosis (MTB) stimulated PBMC in NHS (n ¼ 58) and PTB (n ¼ 48). (A) IFN-g. (B) IL-12p40. (C) IL-4. Note: Detectable IL-4 producers in NHS ¼ 20 out of 58 and PTB ¼ 7 out of 48. Results are shown as mean 7 SEM of both responders and non-responders. (D) IL-5. Table 1 Frequency of HLA-DRB1 alleles in healthy controls and pulmonary tuberculosis patients. HLA-DRB1 Allele

Controls (n ¼ 58) %PF

Patients (n ¼ 48) %PF

DRB1*01 DRB1*15 DRB1*16 DRB1*03 DRB1*04 DRB1*11 DRB1*12 DRB1*13 DRB1*14 DRB1*07 DRB1*08 DRB1*09 DRB1*10

3.4(2) 34.5(20) 1.7(1) 17.2(10) 25.9(15) 13.8(8) 10.3(6) 22.4(13) 12.1(7) 22.4(13) 6.9(4) 0(0) 29.3(17)

4.2(2) 37.5(18) 4.2(2) 18.8(9) 22.9(11) 10.4(5) 12.5(6) 25.0(12) 22.9(11) 25.0(12) 2.1(1) 2.1(1) 12.5(6)

PF, phenotype frequency.

IL-10 level was increased in response to CFA (p ¼ 0.0004) and live M. tuberculosis (p ¼ 0.03) in DRB1*12-positive NHS (n ¼ 6) compared to DRB1*12-negative NHS (n ¼ 52)

(Fig. 3D). A significantly increased IL-6 response was seen in DRB1*04-positive patients (n ¼ 11) upon stimulation with CFA (p ¼ 0.01) and live M. tuberculosis (p ¼ 0.02) compared to DRB1*04-negative patients (n ¼ 37) (Fig. 3E), while NHS showed no association between HLA-DRB1 alleles and IL-6 response (data not shown).

Discussion The decreased Th1 (IFN-g, IL-12p40) response to live M. tuberculosis and CFA observed in patients may be due to CD4+ T cell apoptosis that is probably caused by overexpression of molecules important in the induction of apoptosis such as FasL, TNF-a, TGF-b, and decreased levels of anti-apoptotic molecule Bcl-2.23 An increased IL-6, IL-8 and TGF-b response observed in patients might lead to decreased production of IFN-g, favouring a preferential Th2 response. Increased IL-6 along with TGF-b and IL-8 responses in patients may cause infiltration of cells and initiation of Th17 pathway leading to elevated inflammatory processes associated with pathogenesis of TB. The HLA/peptide complexes generated in response to infection determine the patterns of cytokines secreted and influence the outcome of the immune response.11 The role

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P. Selvaraj et al. DRB1∗15 POSITIVE PTB DRB1∗03 POSITIVE NHS

50

IFN-GAMMA (ng/ml)

40 35

DRB1∗03 NEGATIVE NHS

30 25

∗∗ (38)

∗ (38)

20 15 10

0

5500

DRB1∗10 POSITIVE NHS

5000

DRB1∗10 NEGATIVE NHS

4500

6 4

∗ (18)

∗∗ (18)

CFA 5000

P=0.02 ∗∗ (17)

4500 4000

3500

∗∗ (41) (17) (41)

DRB1∗12 NEGATIVE NHS P=0.03 ∗∗ (6)

3000 2500 ∗ (52)

2000

1500

1500

1000

1000

500

500

0

MTB

DRB1∗12 POSITIVE NHS

P=0.0004 ∗ (6)

3500 IL-10 (pg/ml)

IL-12p40 (pg/ml)

8

MTB

4000

2000

10

0 CFA

2500

P=0.04 ∗∗ (30)

2

5

3000

P=0.04 ∗ (30)

12

P=0.03 ∗∗ (20)

P=0.01 ∗ (20)

IFN-GAMMA (ng/ml)

45

DRB1∗15 NEGATIVE PTB

14

∗∗ (52)

0 CFA

CFA

MTB 400 350

IL-6 (ng/ml)

300

MTB

DRB1∗04 POSITIVE PTB DRB1∗04 NEGATIVE PTB P=0.01 ∗ (11)

P=0.02 ∗∗ (11)

250 200

∗ (37)

150

∗∗ (37)

100 50 0 CFA

MTB

Fig. 3 Influence of HLA-DRB1 alleles on cytokine response to CFA and live M. tuberculosis in NHS (n ¼ 58) and PTB patients (n ¼ 48). (A) Increased IFN-g in DRB1*03-positive NHS. (B) Decreased IFN-g in DRB1*15-positive PTB patients. (C) Increased IL-12p40 in DRB1*10positive NHS in response to live M. tuberculosis. (D) Increased IL-10 in DRB1*12-positive NHS. (E) Increased IL-6 in DRB1*04-positive PTB patients. Comparison was made between DRB1 allele-positive individuals and respective allele-negative individuals in the same group.

ARTICLE IN PRESS HLA-DR and cytokine profile in tuberculosis of HLA-DR polymorphism in modulating the cytokine profile towards M. leprae heat shock protein has been reported.10 An increased production of IFN-g was observed in HLADRB1*03-positive NHS and a decreased IFN-g production was observed in HLA-DRB1*15-positive patients in response to M. tuberculosis and its antigens. Increased production of IFN-g in response to PPD in HLA-DRB1*03-positive normal individuals and a decreased production of IFN-g in HLA-DRB1*02 normal subjects have been observed in an earlier study.24 Preferential responses towards dominant mycobacterial T cell epitopes in HLA-DRB1*03-positive individuals have been described previously.25 Moreover, a reduced frequency of HLA-DRB1*03 has been observed in TB patients and was associated with unfavourable development of the disease.26 It remains to be determined whether the increased IFN-g response observed in HLA-DRB1*03-positive NHS in the present study might contribute to protection against TB. Decreased IFN-g production in HLA-DRB1*15 patients might be due to the increased suppression of the Th1 response. This finding is supported by the observation that spontaneous IL-10 production is more in HLA-DRB1*15-positive patients compared to HLA-DRB1*15-negative patients. IL-10 is a potent inhibitor of HLA class II expression, antigenspecific proliferation and IFN-g synthesis. Earlier studies have shown that DRB1*15 is associated with susceptibility to TB in the south Indian population.27,28 It has also been shown that patients with HLA-DRB1*15 had higher levels of antibodies to M. tuberculosis and were associated with more severe form of PTB.13,16,26,28 Increased IL-12p40 response to M. tuberculosis was seen in HLA-DRB1*10-positive NHS. In our previous study, a significantly decreased macrophage phagocytosis was observed in DRB1*10-positive healthy subjects (unpublished result). The elevated IL-12p40 might increase the IFN-g response to M. tuberculosis, thereby downregulating receptors involved in phagocytosis of M. tuberculosis.2 An increased IL-10 response was observed in DRB1*12-positive NHS. Since IL-10 downregulates macrophage activation, it will be interesting to study if macrophage activation is suppressed in HLA-DRB1*12-positive NHS. A significantly increased IL-6 response was seen in DRB1*04-positive patients. This suggests that HLA-DRB1*04 allele along with HLA-DR2 or other alleles may augment the IL-6 responses which in turn may upregulate the production of M. tuberculosis-specific antibodies by B cells.29 Our earlier study revealed that HLA-DR2/DR3, DR2/DR4 and DR2/DR5 are the major heterozygous combinations associated with susceptibility to TB.29 The present study revealed the association of HLA-DRB1 alleles and cytokine secretion in response to live M. tuberculosis and its CFA in a comprehensive manner. Although the whole cell and culture filtrate of M. tuberculosis are extremely complex, they were used in the present study because the percentage of responders was high compared to defined major antigens of M. tuberculosis.30 However, many studies with defined M. tuberculosis antigens such as ESAT-6 and Antigen 85B have identified the immunodominant peptides that induce IFN-g and they are frequently recognized by most of the patients in the context of multiple HLA-DR alleles.31,32 Hence, further studies with defined M. tuberculosis antigens or peptides may help to identify antigens or peptides that may induce IFN-g in the

549 context of multiple HLA-DR alleles present in the study population and would be of relevance for diagnosis and developing new generation vaccines. Other HLA genes that are in linkage disequilibrium with specific HLA-DRB1 alleles and single nucleotide polymorphisms in the cytokine genes may also influence cytokine secretion. The present study suggests that HLA-DRB1 alleles influence cytokine response to M. tuberculosis antigens, which in turn play a major role in immunity to TB. Funding: Senior Research Fellowship provided to Ms D. Nisha Rajeswari by the Indian Council of Medical Research (ICMR) is acknowledged. Competing interests: None declared Ethical approval: The study was approved by the institutional ethical committee.

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