Plasma levels of Galectin-9 reflect disease severity in malaria infection

Malaria infection is still a big challenge for public health; around 214 million new cases of malaria worldwide occurred in 2015 (range 149–303 million) [1]. Plamosdium falciparum is responsible for the most severe cases of malaria and the greatest number of deaths. Detection and monitoring of severe cases are remaining issues for patient treatment [2].

Galectin-9 (Gal-9) is member of the galectin family of β-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD) [3]. Gal-9 was first described as an eosinophilic chemoattractant [4]. Gal-9 is presumed to bind a variety of molecules including glycoproteins and glycolipids of immune cells and pathogens [5]. These binding properties exert a variety of physiological and pathological functions such as cell differentiation, adhesion, aggregation, and cell death [6]. The elevation of plasma Gal-9 levels during acute human immunodeficiency virus (HIV) infection followed by a rapid decrease after anti-retroviral therapy was reported [7, 8]. Increase in plasma Gal-9 has also been seen during acute dengue virus infection [9]. Gal-9 inhibits production of several pro-inflammatory cytokines (TNF, IL-6, IL-1α) and enhances production of IL-10 [10]. These cytokines are believed to be involved in pathophysiology of malaria [11‐13]. Recent findings of co-inhibitory or immune checkpoint receptors have been giving a novel insight into immune regulation. Because these receptors have a critical role in the maintenance of immune homeostasis: their expression on effector T cells ensures the proper contraction of effector T cells responses and their expression on regulatory T (Treg) cells guarantees the proper function of Treg cells to control effector T cells [14, 15].

It has been reported that PD1, a co-inhibitory receptor play a major role for inactivating a protective immunity in malaria infection in mice [16]. Gal-9 binds to T cell immunoglobulin and domain 3 (Tim-3) a co-inhibitory receptor, and that binding can ameliorate experimental autoimmune encephalomyelitis (EAE) by inducing cell death in Tim-3⁺ Th1 cells [17, 18]. Gal-9 also regulates activated CD8+ T cells [19], which increase during malaria [20]. Activation of CD8+ T cells these cells is considered as a risk factor of anemia in severe malarial [21]. These emerging regulatory activities of Gal-9 involving co-inhibitory receptors took us to investigate Gal-9 in malaria, though how Tim-3 functions to determine effector T cell responses is not yet well clarified [15]. Recently expression of Gal-9 and Tim-3 was demonstrated in lung, mediastinal lymph nodes and liver tissues in murine malaria model [22, 23]; however, the plasma levels of Gal-9 in human malaria patients has not been evaluated yet.

In this study, the plasma levels of Gal-9 in malaria patients were measured at three time points (days 0, 7 and 28) to examine its kinetics and association with cytokines, chemokines and clinical parameters.

This study revealed the presence of Gal-9 in plasma in malaria patients and showed that plasma levels of Gal-9 accurately reflect the status of inflammation and recovery during malaria infection for the first time though the tissue expression of Gal-9 and Tim-3 was demonstrated in murine malaria model [21, 22]. The elevation of Gal-9 was reported in other acute and non-acute infectious diseases such as dengue virus infection (median level was 1525 pg/mL), leptospirosis (616 pg/mL) [9], acute HIV infection (~2300 pg/mL) [8] and active tuberculosis (200 pg/mL) [25]. Detailed analysis with clinical parameters in dengue virus infections showed its association with the clinical severity. Gal-9 exerts its pivotal immunomodulatory effects by inducing apoptosis or suppressing effector functions via engagement with its receptor, Tim-3 a co-inhibitory receptor, though how Tim-3 functions to determine effector responses have not been clarified, yet [14, 15]. The versatile activities of Gal-9 was attributed by dual activities of the molecule, anti-microbial as well as anti-inflammatory activities [26].

It has been suggested that Gal-9 production might be stimulated by the response of mounting Th1 cells that occurs during P. falciparum infection, and produced Gal-9 was assumed to inhibit their activation [10, 14]. This may explain the correlation seen in this study between Gal-9 and inflammatory cytokines (IFN-γ, TNF, IFN-α and IL-6) and chemokines (MIP-1β, MCP-1 and Fractalkine) at day 0. It was proposed that the levels of TNF and IL-6 are indicators of malaria severity [11]. This association of these molecules with Gal-9 in both UM and SM cases supports the idea of Gal-9 as a severity marker in malaria infection. In fact, Gal-9 levels correlated positively with kidney parameters and inversely with electrolytes.

The correlation of Gal-9 with IL-10 could be related to the effect of Gal-9 on differentiation of naïve T cells to Gal-9⁺ ThGal and Treg, which express high levels of IL-10 mRNA [27]. Though both Gal-9 and IL-10 were proposed to have anti-inflammatory properties, it is known that IL-10 production is impaired in SM compared with UM as reported elsewhere [28]. Another anti-inflammatory cytokine IL-1Ra levels correlated with Gal-9 both in UM and SM cases and IL-1Ra was found to be correlated with parasitemia [29]. In malaria, a variety immune cells are known to be activated such as CD8+ T, CD4+ T and NK cells [30‐32], and their function can be regulated or impaired by Gal-9 in vitro [19, 33]. However, it is difficult to demonstrate if the released Gal-9 could constrain immune response through the Gal-9/Tim-3 pathway [19]. Gal-9 shares the receptor Tim-3 with high mobility box group-1 (HMBG-1), an inflammation mediator [14, 34]. HMGB-1 elevation was reported an as informative prognostic marker for disease severity in human severe malaria [35]. In contrast to the apoptotic effect of Gal-9 on T cells [14], HMGB-1 has been described as having proliferation effects by binding through HMGB-1/Tim-3 and HMGB-1/RAGE on T cells [36, 37]. The differential roles of these two ligands (HMGB-1 and Gal-9) in controlling the immune response against parasites should be clarified in future studies.

Beside immune modulatory activities, Gal-9 is released from infected or damaged cells, and could act as danger signal molecules [9], by initiating inflammation as reported in murine respiratory tularaemia [38]. Moreover the Tim-3-Gal-9 expression was found to be elevated in acute lung injury in a murine malarial model [22].

The released Gal-9 can also bind to galactosides of pathogens, pathogen-associated molecular pattern (PAMP), as pathogen recognition receptors (PRRs), as shown recognition of the Leishmania major Poly-β-galactosyl epitope by Gal-9 [39]. Future Investigation on the biological roles of Gal-9 in malaria infection would be necessary.

Taken together, this study shows an increase of plasma Gal-9 levels during malaria infection, which is capable of identifying severe cases, and is tracking the inflammation process. Therefore, it may be used as a novel biomarker of malaria infection.