Functional role of galectin-9 in directing human innate immune reactions to Gram-negative bacteria and T cell apoptosis

Highlights

• Galectin-9 is involved in the opsonisation of Gram-negative bacteria.

• Galectin-9 promotes anti-bacterial immune defence.

• Galectin-9 suppresses cytotoxic T cell function.

• This effect can be achieved by “opsonisation” of T cells by galectin-9.

• Affected apoptotic T cells can be phagocytosed by macrophages.

Abstract

Galectin-9 is a member of the galectin family of proteins, which were first identified to specifically bind to carbohydrates containing β-galactosides. Galectin-9 is conserved through evolution and recent evidence demonstrated its involvement in innate immune reactions to bacterial infections as well as the suppression of cytotoxic immune responses of T and natural killer cells. However, the molecular mechanisms underlying such differential immunological functions of galectin-9 remain largely unknown. In this work we confirmed that soluble galectin-9 derived from macrophages binds to Gram-negative bacteria by interacting with lipopolysaccharide (LPS), which forms their cell wall. This opsonisation effect most likely interferes with the mobility of bacteria leading to their phagocytosis by innate immune cells. Galectin-9-dependent opsonisation also promotes the innate immune reactions of macrophages to these bacteria and significantly enhances the production of pro-inflammatory cytokines – interleukin (IL) 6, IL-1β and tumour necrosis factor alpha (TNF-α). In contrast, galectin-9 did not bind peptidoglycan (PGN), which forms the cell wall of Gram-positive bacteria. Moreover, galectin-9 associated with cellular surfaces (studied in primary human embryonic cells) was not involved in the interaction with bacteria or bacterial colonisation. However, galectin-9 expressed on the surface of primary human embryonic cells, as well as soluble forms of galectin-9, were able to target T lymphocytes and caused apoptosis in T cells expressing granzyme B. Furthermore, “opsonisation” of T cells by galectin-9 led to the translocation of phosphatidylserine onto the cell surface and subsequent phagocytosis by macrophages through Tim-3, the receptor, which recognises both galectin-9 and phosphatidylserine as ligands.

Introduction

Galectin-9 is a member of the galectin family of proteins which were first identified to specifically bind to carbohydrates containing β-galactosides [1], [2], [3], [4], [5]. Galectins vary in their structural organisation and, so far, three different forms of galectin structure were discovered. Galectins can display dimeric, chimeric or tandem structures [1], [2], [3]. Galectin-9 has a tandem structure and contains two distinct carbohydrate recognition domains (CRDs) within one polypeptide [1], [2], [3], [4], [5]. The CRDs are fused together by a peptide linker. Galectin-9 may be present in three main isoforms characterised by the length of their linker peptide which can be long (49 amino acids), medium (27 amino acids) and short (15 amino acids) [1], [2], [3], [4], [5].

Galectins are conserved through evolution and have various intracellular and extracellular functions including both normal and pathophysiological processes [1], [2]. Galectin-9 is one of the most important galectins and is a major contributor to human immune reactions [6], [7], particularly because of its ability to suppress the cytotoxic activities of T and natural killer (NK) cells. In cytotoxic T cells galectin-9 acts through receptors such as Tim-3 (T cell immunoglobulin and mucin-containing protein 3) and VISTA (V-domain Ig-containing suppressor of T cell activation) [7]. Galectin-9 can induce leakage of granzyme B proteolytic enzyme from the intracellular granules of cytotoxic T cells thus leading to their programmed death [7]. In NK cells, galectin-9 acts mainly through Tim-3 and impairs their cytotoxic activities [6]. As such, galectin-9 is used by cancer cells to escape immune surveillance and also by foetus cells where it protects the embryo against rejection by the mother’s immune system [8]. Furthermore, galectin-9 was found to participate in neutrophil-mediated killing of Gram-negative bacteria by opsonisation, thus promoting their phagocytosis by neutrophils [9].

However, the actual biochemical role of galectin-9 in anti-bacterial immune defence and suppression of T cell functions remains to be comprehensively understood. Here we report that galectin-9 binds Gram-negative bacteria (E. Coli XL-10 Gold) by interacting with lipopolysaccharide (LPS), which is a crucial cell wall component. This opsonisation effect renders the bacteria less mobile thus facilitating their capture and phagocytosis by macrophages. Opsonisation also promotes the innate immune reactions of macrophages to Gram-negative bacteria and significantly enhances the production of pro-inflammatory cytokines – interleukin (IL) 6, IL-1β and tumour necrosis factor alpha (TNF-α). Galectin-9 was almost incapable of binding peptidoglycan (PGN), which forms the cell wall of Gram-positive bacteria. Galectin-9 associated with the cell surface (studied in primary human embryonic cells) was not involved in the interaction with bacteria or bacterial colonisation. However, cell-surface-based galectin-9 on human embryonic cells, as well as secreted galectin-9, targeted T lymphocytes and caused apoptosis in T cells expressing granzyme B. T cells “opsonised” by galectin-9 were phagocytosed by macrophages through Tim-3. Furthermore, galectin-9 induced the release of transforming growth factor beta type 1 (TGF-β) and high mobility group box 1 (HMGB1) from T cells. TGF-β induces the expression of galectin-9 in cancer and embryonic cells and HMGB1 enhances the ability of macrophages to phagocyte apoptotic T cells.

Taken together our results suggest that galectin-9 is capable of opsonising LPS-containing bacteria and T cells triggering their phagocytosis by macrophages. Moreover, galectin-9 provokes the activation of anti-bacterial innate immune reactions and, in the case of T cell suppression, indirectly enhances the phagocytic activity of macrophages.