Increased expression of histocompatibility leukocyte antigen-G in the tuberculoid form of leprosy

1. Introduction

Leprosy, caused by Mycobacterium leprae (M. leprae) and Mycobacterium lepromatosis (M. lepromatosis),¹ is a chronic neurodermatological disease that affects approximately 180,000 people globally each year. Brazil reported 22,773 cases by the end of 2023, the second-highest number worldwide.² The disease is classified according to the clinical, histological, and cellular immune response.³ Tuberculoid leprosy (TT) involves an efficient cell-mediated immune response (CMI) by Th1 cells, which limits bacillary multiplication. This form often presents with loss of sensation and early peripheral nerve involvement, causing irreversible damage. Lepromatous leprosy (LL) lacks CMI, resulting in high bacillary load, high levels of specific antibodies, and widespread skin lesions. Borderline leprosy, an unstable form, can be classified based on CMI level response into borderline-tuberculoid (BT), borderline-borderline (BB), and borderline-lepromatous (BL).⁴ Moreover, leprosy can be further classified based on bacilloscopy and the number of lesions, as recommended by WHO.⁵

Leprosy reactions are immunological complications occurring in 30% to 50% of patients before, during, or after multidrug therapy and often require medical intervention.⁶ Type 1 reaction or reversal reaction (RR) is mediated by T-cells specific against M. leprae antigens due to increasing CMI, which occurs in BT, BB, and BL patients.⁷ Type 2 reaction, or erythema nodosum leprosum (ENL), is immune complex-mediated, resulting in inflammation and leukocyte chemotaxis.⁷ It affects BL and LL patients with poor CMI to M. leprae, abundant bacilli, and a strong humoral response. Both reactions can cause peripheral nerve damage, leading to sensory loss, paralysis, and deformity, resulting in significant functional morbidity.⁷

The human leukocyte antigen-G (HLA-G) is an immunoregulatory molecule identified in maternal-foetal immune tolerance.⁸ This molecule interacts with various inhibitory receptors, including immunoglobulin-like transcript 2 (ILT2), ILT4, and killer immunoglobulin-like receptor 2 (KIR2DL4).⁹ The binding of HLA-G to these receptors can trigger several immunological events, such as apoptosis, and block the cytotoxic activity of CD8+ T lymphocytes; prevent CD4+ lymphocyte proliferation; inhibit antigen presentation and differentiation of B-lymphocytes; modulating the NK cells and DCs activity; regulation of Th1/Th2 cytokines balance favouring Th2 profile, and induction of regulatory T-cells (Tregs) expansion.⁹

In physiological conditions, HLA-G expression is limited to a few tissues.¹⁰ However, in some diseases, there is considerable variation in HLA-G levels.^11-16 This can modulate the immune response, promoting immune escape in the context of infectious diseases and tumours.¹⁷ In leprosy, Silva et al. observed a higher level of HLA-G expression in skin lesions of PB leprosy patients in the Middle-West region of Brazil.¹⁸ Lucena-Silva et al. found that allele A of the polymorphism +3187A/G in the HLA-G gene had a protective effect against multibacillary (MB) leprosy. However, it has been linked to ENL in the Northeast region of Brazil.¹⁹ This study investigated HLA-G expression in different clinical forms of leprosy and leprosy reactions in the Middle-West region of Brazil, an endemic area for the disease, to understand the role of these molecules in leprosy and confirm previous findings in the Brazilian population.

Methods

Results

HLA-G expression is more prevalent in patients with PB leprosy skin lesions

HLA-G expression was found in 75.0% (18/24) of paucibacillary (PB) leprosy patients, while it was seen in only 36.17% (17/47) MB patients. The expression was observed in well-formed epithelioid granulomatous infiltrates localised in the dermis, with macrophages showing cytoplasmic and nuclear HLA-G positivity [Figure 1]. A significant association was observed between HLA-G expression and PB leprosy (OR= 5.29; p<0.003; CI: 1.76-15.9, Fisher’s exact test).

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Figure 1:

HLA-G Expression in Leprosy Patients and Controls. (a) PB leprosy: well-formed epithelioid granulomatous infiltrates localised in the dermis, with macrophages showing positive HLA-G cytoplasmic and nuclear expression. (b) MB leprosy: disorganised aggregates of foamy histiocytes, with occasional lymphocytes and negative HLA-G expression. (c) Normal skin with no HLA-G expression.

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The concentration of sHLA-G in the serum does not vary between different clinical forms of leprosy or leprosy reactions

Soluble HLA-G was detected in all the clinical manifestations of leprosy, leprosy reactions, and healthy controls [Figure 2]. The concentration of sHLA-G ranged from 24.72 to 1035.2 units/mL (mean: 197.6 units/mL) in TT; 28.64 to 488.8 units/mL (mean: 219.0 units/mL) in BT; 36.16 to 135.2 units/mL (mean: 75.81 units/mL) in BB; 16.16 to 52.32 units/mL (mean 34.44 units/mL) in BL; and 29.52 to 64.24 units/mL (mean 44.56 units/mL) in LL. In patients with leprosy reactions, the concentration of sHLA-G ranged from 11.52 to 81.2 units/mL (mean 53.65 units/mL) in RR and 32.48 to 422.64 units/mL (mean 154.32 units/mL) in ENL. In healthy controls, sHLA-G concentrations ranged from 14.32 to 159.44 units/mL, with a mean value of 62.58 units/mL. No significant differences were found between patients and healthy controls or different clinical forms of leprosy (p = 0.372), and no correlation was observed between sHLA-G levels and in situ HLA-G expression (p = 0.641) [Figure 2].

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Figure 2:

Soluble HLA-G concentration in the clinical forms (TT, BT, BB, BL, LL, RR, ENL reactions, and controls). CTRL: Healthy controls. Symbols representing clinical forms: (●TT: black circle), (■BT: black square), (▲BB: black upward triangle); (▼BL: black downward triangle), (♦LL: black diamond), (○ENL: white circle with black outline), (□RR: white square with black outline), (△CTRL: white upward triangle with black outline)

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Discussion

The study of HLA-G is based on the premise that this molecule influences the effectiveness of the immune response through a range of interactions with various cell types. HLA-G has been shown to reduce the efficacy of immune responses in infectious diseases, thereby aiding pathogens in evading the immune system.²⁰ This study assessed HLA-G expression in patients with different manifestations of leprosy recruited from a leprosy-endemic region.

HLA-G expression has been observed in all clinical forms of leprosy, with PB patients exhibiting higher levels of expression than those in the MB group. TT patients demonstrated higher HLA-G expression levels than patients with other clinical forms and healthy controls, corroborating the results observed in the PB group. The findings are consistent with those reported by da Silva et al.,¹⁸ who also observed elevated HLA-G expression in the aforementioned group. A gradual decline in the HLA-G expression was observed across clinical forms, with LL patients exhibiting lower amounts than TT patients. These results are consistent with those reported by Lucena-Silva et al., who associated the +3187A allele with decreased mRNA stability and low HLA-G production in vitro with MB leprosy.¹⁹

Th1 immune responses are characteristic of PB leprosy (TT and BT), while in MB leprosy (BB, BL, and LL), the Th2 immune response is prevalent.^4,21 Research hitherto demonstrated that both IFN-γ associated with the Th1 profile and IL-10, linked to the Th2 profile, induce HLA-G expression and modulate its levels.¹⁷ Conversely, HLA-G activation has been shown to induce an imbalance in cytokine profiles, favouring a Th2 shift over a Th1/Th17 profile. The induction of HLA-G expression by cytokines that exhibit antagonistic properties, such as IL-10 and IFN-gamma, may be associated with distinct phases of the immune response.²¹ Therefore, the induction of HLA-G by IFN-γ in TT may offer protection against tissue damage derived from the intense inflammatory response mediated by this cytokine in the context of a well-established Th1 response. Like leprosy, Saurabh et al.²² reported an increase in the HLA-G expression in localised tuberculosis (TB), which presented with better CMI compared to disseminated disease.

Moreover, according to the biological function of HLA-G, it can be hypothesised that, in addition to HLA-G molecules, their receptors are pivotal in determining the role of this component in the immune response.⁹ In this regard, Saurabh et al.²² observed a decrease in the expression of the ILT-2, an HLA-G receptor, in the localised form of TB compared with the disseminated form. Patients with a more robust CMI may express higher levels of HLA-G due to IFN-γ stimulation but show lower levels of receptors such as ILT-2, ILT-4, or KIR2DL4. In contrast, patients with a more pronounced Th2 response may manifest reduced HLA-G expression, which could be linked to higher expression of receptors like ILT-2, ILT-4, or KIR2DL4, triggering the suppressive phenotype typical of this clinical form. It is conceivable that a comparable mechanism could occur in leprosy. However, further research is needed to confirm this hypothesis.

HLA-G expression does not influence the manifestation of RR and ENL, which are immunologically mediated phenomena. This is supported by the finding that HLA-G expression in patients with leprosy reactions was similar to that in those without. The +3187AA genotype in the HLA-G gene has previously been linked to the manifestation of ENL.¹⁹ Yang and Khoury suggested that factors such as high bacillary exposure, elevated prevalence rates, and increased transmission of leprosy may obscure genetic effects in endemic areas, and the disease risk is a sum of genotype and environmental exposure.²³ This could help explain the lack of association between in situ HLA-G expression and the occurrence of leprosy reactions.

Levels of sHLA-G did not differ between clinical forms, leprosy reactions, and healthy individuals. Again, we expected a higher concentration of sHLA-G in MB patients since elevated levels of IL-10 and TGF-β characterise this clinical form.⁴ These cytokines can be secreted by Treg cells, which also express HLA-G and are known to suppress effector T cells, thereby regulating the immune system.²⁴ Saini et al. reported that TGF-β-secreting FOXP3+ Tregs cells induce an anergic state in T-cells in LL leprosy.²⁴ FOXP3+ Tregs control immune responses to intracellular pathogens responses, such as Leishmania and Mycobacterium tuberculosis.^25,26 Da Silva et al. found higher levels of sHLA-G in healthy individuals, attributing this to regulatory CD4+ T-cells (Tregs), which help balance pro- and anti-inflammatory responses in defence against pathogens.¹⁸

Saurabh et al.²² described a higher concentration of sHLA-G in patients with disseminated TB, linking the increase in the soluble form to the induction of FOXP3-Tregs and elevated levels of IL-10, which contribute to disease dissemination. However, this phenomenon was not observed in leprosy. While HLA-G expression in situ increased in PB patients, higher levels of soluble HLA-G were not observed in this group. This discrepancy may be related to the limited number of lesions observed in PB patients, which may not be sufficient to reflect soluble HLA-G levels in serum.

Limitations

This study was limited by the sample size, particularly the number of patients with ENL forms, which may have impacted the findings. Furthermore, the number of patients available to investigate soluble HLA-G (sHLA-G) limited this analysis. The study did not investigate potential interactions between HLA-G expression and other immune molecules, and changes in HLA-G expression and sHLA-G levels over time.

Conclusion

In conclusion, our findings demonstrate HLA-G expression across the clinical spectrum of leprosy and leprosy reactions, with higher expression observed in TT leprosy patients, confirming previous findings in a Brazilian population. However, no differences were noted in the levels of sHLA-G. The expression of HLA-G in the different clinical forms of leprosy suggests the involvement of these molecules in regulating inflammatory processes. Further studies investigating the expression of HLA-G receptors and their interactions with HLA-G molecules will enhance our understanding of the mechanisms underlying leprosy pathogenesis.