By Katherine Edwards, PhD Student, Queen’s University Belfast
Cytokines play a vital role in immune responses enabling cross-talk between different cell types and inducing activation, differentiation, proliferation and cell migration by altering gene expression in target cell types. There are a wide variety of cytokines each with varying roles which when produced in different combinations can lead to various outcomes. Cytokines can promote inflammation and aid migration of effector immune cells to sites of infection or damage where these cells then work as a team to combat the problem. Cytokines can also be anti-inflammatory – these cytokines are released when the cause of inflammation has been resolved and they alter the phenotype of immune cells to prevent excessive inflammation which can cause unnecessary damage. Excessive inflammation through cytokine signalling can also lead to disease and therefore, it is of great importance to have regulatory systems in place to tightly control cytokine secretion and immune responses.
Following binding of cytokines to their cognate cell surface receptors a signalling cascade is induced. Primarily, the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is utilised by cytokines (Fig. 1). It is here that the family of suppressor of cytokine signalling (SOCS) proteins play their crucial role.
Figure 1 | JAK-STAT activation and induction of gene transcription following cytokine stimulation. Cytokine receptors dimerise upon cytokine binding allowing receptor-associated JAKs to activate one another through trans-phosphorylation. JAKs then phosphorylate tyrosine residues located on the cytoplasmic regions of the cytokine receptors. STAT proteins dock onto these phosphorylated regions permitting them to dimerise through JAK-mediated phosphorylation. Dimerised STATS translocate to the nucleus where they bind specific gene regions resulting in activation or repression of target genes (Adapted from Palmer and Restifo, 2009).
SOCS proteins comprise a family of 8 proteins; SOCS1-7 and cytokine inducible Src homology 2 (SH2)-domain-containing protein (CIS). Their expression is induced by STAT proteins following cytokine-stimulated JAK-STAT pathway activation. SOCS proteins play a vital role in the regulation of cytokine signalling by providing a classical negative feedback loop limiting their production. The SOCS protein structural characteristics play a key role in how they exert their effects on cytokine signalling.
The SOCS proteins share a common structure consisting of an N-terminal region that varies in length, SH2-domain and C-terminal SOCS box motif (Fig. 2).
Figure 2 | The molecular structure of SOCS proteins. All SOCS proteins contain an N-terminus of varying length, a conserved SH2 domain and a highly conserved C-terminal SOCS box. SOCS1 and SOCS3 also contain a kinase inhibitory region (KIR) (Adapted from Elliott and Johnston, 2004).
The SH2 domain binds to phosphotyrosine residues present on the target of the SOCS protein. Following this, the SOCS box interacts with elongin B and C, cullin 5, RING-box-2 and an E2 ubiquitin-conjugating enzyme to form an E3 ubiquitin ligase complex (Yoshimura et al., 2007). The E3 ligase then polyubiquitinates lysine residues within the substrate to target it for proteasomal degradation.
There are three main ways in which SOCS proteins inhibit cytokine signalling. All SOCS proteins have the ability to ubiquinate their targets to mark them for proteasomal degradation. SOCS2, SOCS3 and CIS also have the ability to competitively bind to phosphotyrosine residues on the cytoplasmic regions of cytokine receptors, preventing access of STAT proteins (Trengove and Ward, 2013). SOCS1 and SOCS3 also contain a KIR that allows them to inhibit JAK proteins by acting as a pseudosubstrate, preventing substrate access to the JAK catalytic pocket (Zhou et al., 2017). SOCS7 has also been demonstrated to prevent nuclear translocation of STAT3 and STAT5 phosphorylated dimers (Martens et al., 2005). Thus, SOCS have acquired a number of processes to alter cytokine signalling.
Manipulation of the immune response
Many studies have improved our understanding of how SOCS proteins regulate or alter immune responses. The creation of knockout (KO), and selective KO, mouse models has greatly advanced this field of research. Complete SOCS1 KO and SOCS3 KO cause mice to die at neonatal and embryonic stages, respectively (Roberts et al., 2001; Starr et al., 1998). This demonstrated SOCS1 is required to regulate cell development and postnatal growth, and SOCS3 is required for placental development. SOCS2 KO mice present with gigantism demonstrating SOCS2 is a regulator of growth hormone signalling during development (Metcalf et al., 2000). Alongside these key roles for the SOCS proteins, they have also been demonstrated to have effects on key immune cells and how the immune response is shaped.
Immune cell polarisation
Macrophages are one of the key innate immune cells that mediate immune responses to pathogens, inflammation and tissue repair. Macrophages constantly sample their environment so they can react and alter their phenotype appropriately. SOCS proteins have the ability to polarise macrophages to specific phenotypes, thus shaping the immune response. The classically activated macrophage (M1) is induced by pathogens and results in increased bactericidal and tumouricidal properties by producing pro-inflammatory mediators such as IL-1, -6 and interferon-γ (Ley, 2017). SOCS1 controls this phenotype by suppressing the inflammatory signalling axis, resulting in an anti-inflammatory alternatively activated (M2) phenotype (Elliott and Johnston, 2004; Wilson, 2014). M2 macrophages are induced by IL-4 and IL-10, and produce IL-10 and soluble growth factors, like transforming growth factor-β, to promote tissue regeneration and wound repair (Martinez and Gordon, 2014). They also recruit T helper 2 (Th2) and T regulatory cells to the site of damage/infection to aid immunosuppression, allowing for repair and restoration of tissue homeostasis (Murray and Wynn, 2011). SOCS3, however, has been shown to drive and maintain the M1 phenotype by enhancing nuclear factor-κB activity (Arnold et al., 2014; Liu et al., 2008). SOCS2 was also found to target SOCS3 for proteasomal degradation to limit and shift the M1 phenotype to M2 (Tannahill et al., 2005).
SOCS proteins have also been implicated in T cell responses. Egwuagu et al., (2002) demonstrated SOCS proteins are expressed differentially in T cell sub-types, suggesting a role for SOCS proteins in polarising T cell responses. For instance, SOCS3 is expressed by Th2 cells and positively contributes to their development by enhancing production of the Th2-promoting cytokine IL-4 (Seki et al., 2003).
SOCS and Cancer
SOCS proteins have been implicated greatly in cancer but conflicting results regarding their roles emphasise the need for further research. SOCS1 deletion in macrophages causes a shift to the inflammatory M1 phenotype which leads to anti-tumour activity, thus preventing tumour growth (Hashimoto et al., 2009; Wilson, 2014). Deletion in CD8+ T cells also induces stronger anti-tumour activity (Chikuma et al., 2017). Alternatively, Jiang et al., (2017) found SOCS1 displayed anti-tumour activity by inhibiting tumour proliferation and invasion.
SOCS3 exerts anti-tumour activity and when suppressed in various cancers tumour cell growth occurs consequently (Chikuma et al., 2017; Jiang et al., 2017). While SOCS3 can inhibit anti-inflammatory cytokine production, it cannot inhibit STAT3 activation in the IL-10 pathway (Mahony et al., 2016). Hiwatashi et al., (2011) discovered that inhibiting SOCS3 resulted in prolonged survival of tumour-bearing mice and reduced metastasis. This was due to preventing the inflammatory effects of SOCS3 but not STAT3 activation, which lead to higher expression of the anti-tumour monocyte-chemoattractant protein 2/CC chemokine ligand 8.
The expression and effects of the SOCS proteins have been investigated in many different disease settings and from these findings it can be concluded that altering their expression therapeutically must be performed with great care due to their various mechanisms of activation/suppression and effects in different cell types. Ideally SOCS inhibitors should be cell-targeting and membrane-permeable so they are able to penetrate the cell and bind intracellularly expressed SOCS proteins (Chikuma et al., 2017).
The important immunomodulatory role the SOCS proteins play in the innate and adaptive immune response makes them an attractive therapeutic target for a wide array of inflammatory disorders.
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