Galectin-1 induces 12/15-lipoxygenase expression in murine macrophages and favors their conversion toward a pro-resolving phenotype

Highlights

• The impact of Galectin-1 on macrophages pro-resolving properties was examined in vivo and ex vivo.

• Galectin-1 favored macrophage conversion to a CD11b^low phenotype.

• Galectin-1 enhanced macrophage expression and activity of 12/15-lipoxygenase.

• Galectin-1 enhanced loss of macrophage phagocytic activity (efferocytic satiation).

• Galectin-1 reduced cytokine secretion from LPS-stimulated macrophages.

Abstract

During the resolution of inflammation macrophages undergo functional changes upon exposure to pro-resolving agents in their microenvironment. Primarily, engulfment of apoptotic polymorphonuclear (PMN) cells promotes conversion of macrophages toward a pro-resolving phenotype characterized by reduced CD11b expression. These macrophages are not phagocytic, do not respond to TLR ligands, and express relatively high levels of the pro-resolving enzyme 12/15-lipoxygenase (LO). Here, we report that the immuno-regulatory lectin galectin-1 is selectively expressed by CD11b^high, but not CD11b^low macrophages. Upon exposure in vivo and ex vivo, galectin-1 directly promoted macrophage conversion from a CD11b^high to a CD11b^low phenotype and up-regulated the expression and activity of 12/15-LO. Moreover, galectin-1 treatment in vivo promoted the loss of phagocytic capacity (efferocytic satiation) in peritoneal macrophages and down-regulated secretion of TNF-α, IL-1β, and IL-10 upon LPS exposure. Our results suggest that galectin-1 could be an essential mediator in the control of macrophage function during the resolution of inflammation.

Introduction

During the active resolution of inflammation [1], [2] several components of the immune response are eliminated [3]. Polymorphonuclear (PMN) cells undergoing apoptosis are cleared by macrophages and other phagocytic cells in a non-phlogistic fashion [4], [5], [6], [7], [8]. Apoptotic cell engulfment by phagocytes (also termed efferocytosis [4]) is mediated by signals that are expressed on the surface of apoptotic cells and their corresponding receptors (reviewed in [5], [6]). Apoptotic cells also serve as resolution cues for macrophages, as their recognition evokes distinct signaling events that block the release of pro-inflammatory mediators and allow further engulfment of apoptotic cells [7], [11]. Release of pro-inflammatory mediators is activated by bacterial moieties, and its down-regulation is termed immune-silencing/reprogramming [8], [9]. Macrophage reprogramming is accompanied by the production of TGF-β and, in some cases, IL-10 [10], [11], [12], [13], cytokines that can promote resolution and wound repair. The engulfment of apoptotic leukocytes by macrophages also leads to inhibition of inducible nitric oxide (NO) synthase (iNOS) expression and stimulates the expression of arginase-1 in the RAW 264 macrophage cell line [14], thereby preventing reactive NO production. In addition, the expression of 15-lipoxygenase (LO)-1, which is involved in the generation of pro-resolving lipid mediators [14], [15], [16], [17], as well as the production of angiogenic growth factors [18] by macrophages are consequent to the uptake of apoptotic cells. Of note, molecular entities that are selectively expressed on apoptotic PMN cooperate with soluble bridging molecules in binding distinct receptors on the surface of macrophages. These receptors mediate both apoptotic cell clearance and immune-silencing in resolution-phase macrophages and other phagocytes [13], [19], [20], [21].

Macrophages are highly diverse cells that adopt various functional phenotypes upon receiving differentiation cues from their surrounding environment [22], [23], [24]. Recent reports indicate that macrophages acquire distinct phenotypes during the resolution of acute inflammation [15], [25]. A typical phenotype, which distinguishes resolution-promoting macrophages, is characterized by low expression of CD11b (CD11b^low) and is generated upon engulfment of apoptotic PMN [15]. CD11b^low macrophages are distinct from either pro-inflammatory, classically-activated (M1) or anti-inflammatory, alternatively-activated (M2) macrophages, since they do not express iNOS or arginase-1 [15]. Macrophage conversion to the CD11b^low phenotype results in significant functional changes in addition to the reduction in surface expression of CD11b and F4/80 [15]. Specifically, CD11b^low macrophages stop producing TNF-α and IL-1β, do not engulf apoptotic PMN (efferocytic satiation), increase the production of TGF-β and the expression of 12/15-LO, and emigrate to lymphatic vessels [15].

Galectin-1 (Gal-1; encoded by Lgals1) belongs to the galectin family of proteins. Members of this lectin family typically act in the extracellular milieu through binding to a myriad of glycosylated receptors on the surface of immune cells [26]. Gal-1 is involved in the regulation of both innate and adaptive immunity. It suppresses antigen presentation and NO production by macrophages [27], [28], and de-activates classically-activated microglia during autoimmune neuroinflammation [33]. It also promotes the termination of adaptive immunity by inhibiting pro-inflammatory cytokine production, dampening cell adhesion and trafficking, selectively deleting T helper (Th)1 and Th-17 cells [29], [30], [31], and promoting IL-10 secretion [32], [33], [34]. Gal-1 also induces the differentiation of tolerogenic dendritic cells and regulatory T cells [31], [35]. Importantly, Gal-1 is highly expressed in macrophages during peritonitis and its expression is associated with successful resolution of inflammation [36], [37].

The present study reveals that Gal-1 is expressed by a distinct macrophage population and contributes to a pro-resolving macrophage phenotype both in vivo and ex vivo. Exposure to Gal-1 promotes macrophage conversion from a CD11b^high to a CD11b^low phenotype characterized by enhanced 12/15-LO expression and activity, reduced efferocytic scores in vivo, and diminished cytokine secretion ex vivo.