Mer receptor tyrosine kinase negatively regulates lipoteichoic acid-induced inflammatory response via PI3K/Akt and SOCS3

Highlights

• LTA that activates TLR2 signaling concomitantly induces activation of MerTK signaling in a time-dependent manner.

• LTA-induced MerTK activation is Gas6-dependent.

• LTA induces activation of Akt and SOCS3 in a MerTK-dependent manner.

• Activation of MerTK signaling negatively regulates TLR2-mediated immune response via PI3K/Akt pathway and SOCS3 protein.

Abstract

Activation of toll-like receptor (TLR) signaling that initiates an innate immune response to pathogens must be strictly regulated to prevent excessive inflammatory damage in the host. Here, we demonstrate that Mer receptor tyrosine kinase (MerTK) is a negative regulatory molecule in the lipoteichoic acid (LTA)-induced inflammatory response. LTA that activated TLR2 signaling concomitantly induced activation of MerTK signaling in RAW264.7 macrophages, including phosphoinositide 3-kinase (PI3K)/Akt and suppressor of cytokine signaling 3 (SOCS3). Moreover, LTA induced MerTK activation in a time-dependent manner, and LTA-induced MerTK activation was dependent on the ligand Gas6. Additionally, pretreatment with a specific Mer-blocking antibody significantly inhibited LTA-induced phosphorylation of MerTK, while further enhancing LTA-induced phosphorylation of IκB-α and NF-κBp65 as well as production of TNF-α and IL-6. Meanwhile, the antibody blockade of MerTK markedly prevented LTA-induced Akt phosphorylation and SOCS3 expression, both of which were crucial for the inhibition of TLR2-mediated immune response. Collectively, these results suggest, for the first time, that MerTK is an intracellular negative feedback regulator that inhibits the inflammatory response of LTA-stimulated macrophages through the PI3K/Akt pathway and SOCS3 protein.

Introduction

The innate immune response to pathogens represents the first line of defence against infectious diseases (Rothlin et al., 2007, Takeda and Akira, 2005). Toll-like receptors (TLRs) are prominently expressed in macrophages and dendritic cells (DCs), and TLRs recognize pathogen-associated molecular patterns (PAMP_S) displayed by invading pathogens, which subsequently drive the host cell innate immune response (Kumar et al., 2011, Takeda and Akira, 2005). Although the innate immune response is required for pathogen elimination, the activation of TLRs is a double-edged sword and must be strictly regulated because unrestrained activation of TLRs can lead to autoimmune and inflammatory diseases (Liew et al., 2005, O'Neill, 2007, Rothlin et al., 2007). Therefore, multiple negative regulatory mechanisms to feed back upon and inhibit TLR signaling at various levels have been developed to maintain this immunological balance (Liew et al., 2005).

Remarkably, recent reports have demonstrated that Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases in DCs induces the expression of suppressor of cytokine signaling-3 (SOCS3) and SOCS1 protein, which act to limit TLR signaling (O'Neill, 2007, Rothlin et al., 2007). The TAM ligands are growth arrest specific gene 6 (Gas6) and Protein S, which bind and activate these receptors (Lemke, 2013). Studies have shown that the phenotypes of Mer^−/− mice and TAM^−/− mice are clearly similar and include polyclonal lymphocyte proliferation with tissue infiltrates, splenomegaly, high-circulating autoantibody titres, and broad spectrum autoimmune diseases (Cohen et al., 2002, Lu and Lemke, 2001, Prasad et al., 2006, Scott et al., 2001). In addition, similar to TAM^−/− mice, Mer^−/− mice were shown to be hypersensitive to lipopolysaccharide (LPS)-induced endotoxic shock as a result of excessive production of TNF-α (Camenisch et al., 1999). Altogether, these data support the significant importance of Mer among TAM receptors expressed in immune cells, such as macrophages, DCs, NK cells, and NKT cells. Recently, it has been reported that Mer receptor tyrosine kinase (MerTK) plays a negative regulatory effect on the LPS-induced inflammatory response (Lee et al., 2012). Particularly, lipoteichoic acid (LTA) and LPS represent two major PAMP molecules embedded in the cell wall of Gram-positive and Gram-negative bacteria and are well-known ligands of TLR2 and TLR4, respectively (Akira et al., 2006). LTA shares many inflammatory properties with LPS and plays a critical role in the pathogenesis of inflammatory response (Matsuguchi et al., 2003). However, whether MerTK functions similarly in the inflammatory response of LTA-stimulated macrophages remains to be elucidated.

Activation of TLR2 turns on multiple intracellular adaptor and signaling proteins, including myeloid differentiation factor 88 (MyD88) and TNF receptor associated factor 6 (TRAF6) (Liew et al., 2005). MyD88 protein is the most crucial adaptor in TLR signaling with the exception of TLR3 (Liew et al., 2005). TRAF6 functions as an ubiquitin ligase and is itself activated by ubiquitination. Subsequently, TRAF6 activates mitogen activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways, which result in the production of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-12 (Hacker et al., 2006). Activation of NF-κB is required for the production of pro-inflammatory cytokines and is a multistep process, involving initial phosphorylation and degradation of IκB, subsequent movement of NF-κB heterodimer from the cytoplasm to the nucleus, and then phosphorylation of the NF-κBp65 subunit (Aderem and Ulevitch, 2000, Graham and Gibson, 2005, Liew et al., 2005). Moreover, recent studies have shown that phosphorylation of the NF-κBp65 subunit is essential for maximal NF-κB activity (Graham and Gibson, 2005, Vermeulen et al., 2003, Yang et al., 2003).

With this in mind, the current study was performed to investigate the expression of proteins related to TLR2 pro-inflammatory signaling and MerTK anti-inflammatory signaling in LTA-stimulated RAW264.7 macrophages. Furthermore, we explored the roles and potential mechanisms of MerTK in the regulation of LTA-induced inflammatory response using a specific Mer-blocking antibody.