Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco, a key control factor of pro-inflammatory signalling

Highlights

• Scavenger receptor Marco is required for rapid phagocytosis of Mycobacterium marinum.

• The initial pro-inflammatory response to M. marinum is dependent on Marco.

• marco knockdown leads to increased M. marinum growth in zebrafish embryos.

Abstract

Scavenger receptors on the cell surface of macrophages play an important role in host defence through their ability to bind microbial ligands and induce phagocytosis. Concurrently, signal transduction pathways are initiated that aid in defence mechanisms against the invading microbe. Here we report on the function of scavenger receptor Marco (Macrophage receptor with collagenous structure) during infection of zebrafish embryos with Mycobacterium marinum, a close relative of M. tuberculosis. Morpholino knockdown demonstrates that Marco is required for the rapid phagocytosis of M. marinum following intravenous infection. Furthermore, gene expression analysis shows that Marco controls the initial transient pro-inflammatory response to M. marinum and remains a determining factor for the immune response signature at later stages of infection. Increased bacterial burden following marco knockdown indicates that this scavenger receptor is important for control of M. marinum growth, likely due to delayed phagocytosis and reduced pro-inflammatory signalling observed under conditions of Marco deficiency.

Introduction

Scavenger receptors (SRs) comprise a large family of transmembrane cell surface glycoproteins and are mainly expressed in macrophages, dendritic cells and endothelial cells (Murphy, 2005). Originally these receptors were characterised by their ability to recognise and internalise modified low-density lipoproteins (LDLs), such as oxidised LDL and acetylated LDL, thereby making these susceptible to degradation (Goldstein et al., 1979). For this reason they have been extensively studied in relation to development of atherosclerosis (Kzhyshkowska et al., 2012). However, SRs also have many other important functions, for example in pathogen clearance, in transport of cargo within the cell, and in lipid transport (Dorrington et al, 2013, Pollock et al, 2010, Xu et al, 1997).

Class A SRs in vertebrates consist of three proteins: SR-AI, SR-AII and MARCO (macrophage receptor with collagenous structure). SR-AI and SR-AII are both encoded by the macrophage scavenger receptor 1 (MSR1) gene (Emi et al., 1993) but differ in structure due to alternative splicing of the gene product. SR-AI contains a C-terminal cysteine-rich domain which is lacking in SR-AII (Krieger and Herz, 1994). The trimeric membrane-bound MARCO protein has short intracellular and transmembrane domains, as well as a large extracellular domain that consists of a spacer domain, a long collagenous domain, and a C-terminal scavenger receptor cysteine-rich domain (SRCR domain V) (Elomaa et al., 1995). In un-inflamed tissues, expression of mouse and human MARCO is restricted to macrophages of the splenic marginal zone, medullary cords of the lymph nodes, and alveolar macrophages of the lung (Elomaa et al, 1995, Kraal et al, 2000, Palecanda et al, 1999). Under inflammatory conditions caused by bacterial infection or lipopolysaccharide (LPS) injection, MARCO expression can be rapidly up-regulated (van der Laan et al, 1997, van der Laan et al, 1999) and its expression profile extends to the splenic macrophages of both red and white pulp, dendritic cells (Granucci et al., 2003), and Kupfer cells in the liver (Kraal et al., 2000). Deficiency of MARCO in mouse models and patients is linked with autoimmune diseases such as systemic lupus erythematosus (SLE), which is most likely due to its role in the binding to and clearance of apoptotic cells, which are considered to be a major source for auto-antigens (Podack et al, 2007, Rogers et al, 2009, Tas et al, 2006, Wermeling et al, 2007). The first indication for a role of MARCO in anti-bacterial defence was that monoclonal antibodies inhibited capturing of heat-killed bacteria by macrophages in the marginal zone areas of the spleen in mice (van der Laan et al., 1999).

Recent findings supporting the immunological role of MARCO in human infections are that single nucleotide polymorphisms (SNPs) in MARCO are associated with increased susceptibility to pulmonary tuberculosis (TB) in humans (Bowdish et al, 2013, Ma et al, 2011). Mycobacterium tuberculosis is the causative agent of TB, a common and frequently lethal infectious disease. After being phagocytosed by macrophages, mycobacteria are capable of inhibiting the phagosome-lysosome fusion by interfering with the Rab-controlled membrane trafficking, allowing them to reside and proliferate in a safe environment (Vergne et al., 2004). Increased phagocytic activity of M. tuberculosis has been observed in mouse macrophages deficient in autophagy-related gene 7 (ATG7), which display higher expression levels of MARCO and MSR1 (Bonilla et al., 2013). MARCO has been shown to assist in the phagocytosis of M. tuberculosis in mice via specific binding to the cell wall glycolipid trehalose 6,69-dimycolate (TDM) of this bacteria and to cooperate with TLR2/CD14 signalling in activating cytokine responses (Bowdish et al., 2009). TDM is capable of inducing many elements of M. tuberculosis pathogenesis and is predicted to be present in M. marinum, a natural fish pathogen and a close relative of M. tuberculosis (Tobin and Ramakrishnan, 2008). M. marinum causes an infection in zebrafish (Danio rerio) that shares pathological hallmarks with human TB disease, including the formation of caseating granulomas and development of latency (Parikka et al, 2012, Swaim et al, 2006).

The early life stages of the zebrafish, which are transparent, have proven extremely useful to study the early events in mycobacteria–host interactions, from phagocytosis to early granuloma formation (Cambier et al, 2014, Clay et al, 2007, Tobin et al, 2010, van der Vaart et al, 2013). We have previously shown that a zebrafish homologue of MARCO is expressed in embryos, downstream of the transcription factor Spi1/Pu.1 that is required for myeloid cell development (Zakrzewska et al., 2010). Furthermore, others have demonstrated expression of marco in macrophages and dendritic cells of adult zebrafish (Wittamer et al., 2011). Here we report on a functional study of the zebrafish marco gene.

We demonstrate that Marco is required for the rapid phagocytosis of M. marinum in zebrafish and is an essential player in the establishment of an initial transient pro-inflammatory response to this pathogen. Furthermore, we show that Marco is important for mycobacterial growth control.