Wnt Signalling Pathway in Immunity

The Wnt signalling pathway is an evolutionarily conserved pathway that plays critical roles in the regulation of determining cell fate, polarity, migration, neural patterning and organogenesis during embryonic development. Components of this signalling pathway were discovered in the 1980’s by a number of researchers working in both mouse and Drosophila models.

The name ‘Wnt’ originated from a combination of the Drosophila segment polarity gene wingless (wg) (Sharma, 1973) and the vertebrate homolog, integrated-1 (int-1) (Nusse et al.,1990).Wg was shown to control segment polarity during Drosophila larval development (Bejsovec, 2013), whilst int-1 was identified as a proto-oncogene activated by mouse mammary tumour virus (MMTV) (Nusse and Varmus, 1982).

In most mammalian genomes, including humans, there are 19 Wnt genes of approximately 350-400 amino acids in length which fall into 12 conserved subfamilies (Croce and McClay, 2008), along with 15 Wnt genes in zebrafish and seven in Drosophila (Swarup and Verheyen, 2012). Interestingly, 11 out of the 12 conserved mammalian subfamilies have been identified in the sea anemone, Nematostella vectensis, a member of the Cnidaria phyla (Rigo-Watermeier et al., 2011). Some of these Wnt genes have also been identified in a number of sponge species, of the phylum Porifera (Windsor and Leys, 2010; Windsor Reid et al., 2018). However, they are yet to be identified in single-celled organisms, suggesting Wnt signalling may have been influential in the evolutionary origin of multicellular organisms (Loh et al., 2016). The extensive and diverse conservation of this pathway emphasises the crucial role that Wnt proteins play in organismal patterning and development in a vast array of species.

The Wnt family consists of a large group of cysteine-rich glycosylated proteins that act in an autocrine (same-cell) or paracrine (locally on neighbouring cells) manner to activate numerous signal transduction cascades. The Wnt pathway was first identified for its role in cancer development (Zhan et al., 2017), and then its function in embryonic development, which was discovered in Drosophila when genetic mutations in the Wnt pathway produced abnormal embryos (Cadigan and Nusse, 1997).These Wnt proteins are the known ligand to the cell surface receptor Frizzled (Fzd), which in turn transfers the signal to the intracellular protein, Dishevelled (Dvl).

The Fzd genes were first identified in Drosophila during a screen for mutations that disrupted epidermal cell polarity in the adult fly (Huang and Klein, 2004). Consequently, the genes for the receptor were later found in diverse metazoans, from sponges to humans, and were shown to encode membrane proteins that function in a number of different signalling pathways (Schenkelaars et al., 2015). The Fzd proteins consist of a family of seven-pass transmembrane receptors, which are evolutionarily related to the G-protein coupled receptor (GPCR) superfamily, and conserved throughout the animal kingdom (Dijksterhuis et al., 2014).

The Wnt pathway can be broadly classified into two main types; canonical or non-canonical, both which are activated by the Wnt ligand binding to the Fzd receptor. In the canonical pathway, Wnt proteins released from or presented on the surface of cells act on target cells by binding to Fzd and the co-receptors; low density lipoprotein (LDL) receptor-related proteins 5 and 6 (LRP5/LRP6) on the cell surface. Upon binding, LRP5/6 forms a complex with the Wnt-bound Fzd receptor triggering the activation of Dvl and disassembles a complex containing Adenomatosis Polyposis Coli (APC), Axin and Glycogen Synthase Kinase 3b (GSK3b) (Stamos and Weis, 2013). In simplified ‘on-off’ states of the Wnt pathway, b-catenin (known as Armadillo in Drosophila), a transcriptional co-regulator, is persistently targeted for protosomal degradation by the APC/Axin/GSK3b complex when the pathway is inactive. However, in the presence of Wnt ligands, when the pathway is active, the degradation of b-catenin is prevented and it is able to translocate freely into the nucleus (Valenta et al., 2012) and interact with the transcription factors, such as T cell factor/lymphoid enhancer factor (TCF/LEF), to initiate the transcription of Wnt target genes, including Axin2 and Lgr5 (Ramakrishnan and Cadigan, 2017).

The non-canonical Wnt pathway encompasses the pathways that do not use b-catenin and TCF/LEF, but instead use other downstream effectors (Figure 2) (Shi et al., 2016). These non-canonical pathways may regulate transcriptional and non-transcriptional cellular responses; however, some functions remain unclear. One of the most well characterised b-catenin-independent pathways of Wnt signalling is the planar cell polarity (PCP) pathway (Gao, 2012).

During this signalling cascade, Fzd receptors activate small GTPases Ras related GTPase (RAC), Ras homolog gene family member A (RhoA) and c-Jun N-terminal kinase (JNK) as downstream effectors that can play important roles in controlling cytoskeletal rearrangements and gene expression (Simons and Mlodzik, 2008). In vertebrates, this pathway regulates cell polarity in morphological processes including neural tube closure and gastrulation (Vladar et al., 2009). Interestingly in Drosophila, PCP signalling does not require Wnt ligands, but is still dependent on the Fzd receptor (Schlessinger et al., 2009) suggesting Fzd can be activated by other ligands.

Additionally, the Wnt/Calcium (Ca 2+) pathway (De, 2011), another b-catenin-independent pathway, is activated by Wnt via Fzd to activate heterotrimeric G proteins via Dvl and stimulate the release of intracellular Ca ²⁺ from the endoplasmic reticulum (ER). Through Dvl and phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP3) is activated leading to the release of Ca 2+ and this intracellular accumulation leads to the activation of a number of calcium sensitive proteins, including calcium/calmodulin-dependent kinase II (CamKII) and Calcineurin (Kühl et al., 2000). CamKII has been shown to activate TGF-b activated kinase (TAK1) which is able to antagonise b-catenin/TCF signalling (Dai et al., 2012).

These changes in Ca 2+ levels have also been shown to activate a number of effectors that are able to regulate the transcription of genes involved in controlling cell fate and migration. In recent years, research has indicated that these non-canonical pathways may play a number of roles in cancer, inflammation and neurodegeneration (Ackers and Malgor, 2018; Corda and Sala, 2017; Inestrosa and Toledo, 2008).

It has been known for many years that the Wnt pathway is critical for cellular development and survival, however, its role immunity is not as well defined. Many of the studies investigating the role of Wnt signalling in immunity have been focused on canonical Wnt signalling (Chae and Bothwell, 2018), involving b-catenin and members of the TCF/LEF family.

In blood and immune cells, Wnt signalling helps control the proliferation of progenitor cells (Staal et al., 2008)and may have some influence on cell-fate decisions in stem cells (Ring et al., 2014), however, this remains controversial. It has also been shown in recent years that Wnt proteins may control some aspects of regulatory T cell activation, effector T cell development in the thymus, B cell development in the bone marrow and dendritic cell maturation (Gattinoni et al., 2010; Ma et al., 2012; Swafford and Manicassamy, 2015; Yu et al., 2008).

A number of studies using gain- and loss-of-function experiments have shown that Wnt/b-catenin signalling is an important regulator of T cell development during various stages of thymocyte differentiation (Grigoryan et al., 2008). Using in vivo transgenesis or recombinant retroviruses to mimic loss of Tcf1, a transcriptional activator of Wnt expressed exclusively by T cells in adult mammals, has provided the strongest evidence that the Wnt signalling pathway is detrimental for T cell development (Yu et al., 2010).

Mice with a deficiently in Tcf1 have impaired maturation of thymocytes with defects at the double-negative (DN) and immature single-positive (ISP) stages of T cell development (Figure 3) (Gegonne et al., 2018).

Both CD4⁺ and CD8⁺ T cell counterparts are able to respond to Wnt signalling and findings indicate that Wnt/b-catenin signalling is functionally important during T cell maturation in vivo (Figure 4) (Gattinoni et al., 2010). Following antigen activation, naïve CD4 ⁺ T cells polarise into T helper (Th) cell populations and secrete a distinct set of cytokines including IFN-g (Th1 cells), IL-4/IL-13 (Th2 cells) and IL-17 (Th17 cells). Typically, CD4 ⁺ T cell polarisation results from diverse transcriptional programs triggered by these cytokine signals (Martinez-Sanchez et al., 2018), however, recent findings have shown that Wnt proteins are also able to influence the lineage choice of these cells (Gattinoni et al., 2010; Staal and Sen, 2008).

The blockade of Wnt/b-catenin signalling by the Wnt antagonist Dickkopf-related protein-1 (Dkk1) or siRNA to mediate the silencing of b-catenin, resulted in reduced expression of the master transcription factor GATA binding protein 3 (Gata3) and low levels of Th2 secreted cytokines, IL-4 and IL-13 (Gattinoni et al., 2010). Furthermore, forcing the expression of stabilised b-catenin enhanced Gata3 transcription and IL-4 production. These data together indicate that Wnt signalling is crucial for the regulation of Th cell, specifically Th2, polarization by promoting Gata3 expression. In CD8⁺ T cells, it has been shown that Wnt/b-catenin signalling can favour CD8 ⁺ T cell memory formation by suppressing their maturation into terminally differentiated effector T cells (T _EFF).

Constitutively activating Wnt signalling, via expression of Tcf1 and b-catenin stabilisation, led to enhanced memory formation and reduced expansion of antigen-specific CD8 ⁺ T cells (Zhao et al., 2010). However, deletion of Tcf1 resulted in decreased central memory T cells (T _CM) and impaired their ability to mediate secondary immune responses to pathogen re-challenge (Zhou and Xue, 2012). Furthermore, the maintenance of long-term CD8⁺ T cell memory was drastically impaired when Tcf1 was absent due to reduced responsiveness to IL-15 and a reduction in expression of the anti-apoptotic molecule, Bcl-2 (Zhou et al., 2010).

Gene analyses of Tcf/Lef have shown Tcf7 and Lef1 are highly expressed by naïve CD8 ⁺ T cells (T_N) but levels drop rapidly after a successful encounter with antigen as they undergo expansion and differentiation into cytotoxic T _EFF (Xing et al., 2016). Additionally, high levels of Tcf7 and Lef1 are also found in T_CM cells but low levels are found in effector memory T cells (T _EM). This tightly regulated expression of these Wnt signalling transducers suggests the pathway in CD8⁺ T cells is associated with the function and maintenance of the less-differentiated cells, such as T _N and T_CM (Gattinoni et al., 2009).

In summary, studies have shown that the Wnt/b-catenin pathway is critical for controlling a number of aspects of CD4 ⁺ T cell development, including lineage fates and T cell survival (). It has also been shown to favour Th2 over Th1 polarisation and can increase the persistence of natural CD25⁺Foxp3⁺ regulatory T cells (nTreg) (van Loosdregt et al., 2013), however other Th subsets need to be further investigated. Findings have also shown that Wnt/b-catenin signalling supports CD8 ⁺ T cell differentiation and memory formation under physiological conditions in vivo(Jeannet et al., 2010).

Little is known about the role of Wnt signalling in B cells development and maintenance. In recent years, it has been shown that soluble Wnt proteins act directly on progenitor B (Pro-B) cells in the bone marrow (Figure 5) to induce entry into cell cycle and to proliferate (Reya et al., 2000). However, mice lacking LEF-1, which forms a complex with b-catenin, have defects in proliferation and survival of these Pro-B cells (Petropoulos et al., 2008; Reya et al., 2000). These data begin to establish a link between Wnt signalling and normal B cell development, but there is currently no evidence to suggest Wnt signalling is required for the differentiation of B cells.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that play essential roles in initiating and regulating adaptive immune responses. Recent studies have shown that DCs express the two Wnt co-receptors, LRP5 and LRP6, and Wnt signalling in DCs plays an important role in regulating a balance between tolerance and inflammatory responses. Wnt proteins are able to condition DCs to a regulatory state (Swafford and Manicassamy, 2015) and Wnt-conditioned DCs are able to promote regulatory T cell responses and suppress disease in experimental models of multiple sclerosis (MS) (Suryawanshi et al., 2015). Studies have highlighted that Wnt signalling, both the canonical and non-canonical pathways, may work as a molecular switch in APCs to dampen down excessive inflammation and may provide some host protection from immune-mediated pathology in MS, inflammatory bowel disease (IBD), lupus and arthritis (MacDonald et al., 2009).

Macrophages are specialised cells that are involved in the detection, removal (via phagocytosis) and destruction of harmful organisms that enter the body. Similarly to DCs, macrophages are also able to present antigens to T cells and initiate an immune response by releasing cytokines to activate other immune cell types. Macrophages infected with Mycobacterium bovis , which can cause Tuberculosis (TB) in humans and cattle,showed a robust increase in Wnt5a and the Notch-target genes; cyclooxygenase 2 (COX-2), prostaglandin E2 (PGE2) and suppressor of cytokine signalling-3 (SOCS-3), which are all involved in the down-modulation of the inflammatory response (Brandenburg and Reiling, 2016). Interestingly, when cancer stem cell (CSC) cultures are infected with Salmonella Typhimurium, there is an upregulation of Fzd receptors 2, 7 and 8 along with Wnt2 and 11, however, when macrophages are infected with Mycobacterium tuberculosis, Fzd4 and Wnt5a are highly expressed (Villaseñor et al., 2017).

These observations indicate that specific sets of Fzd receptors and Wnt ligands are expressed depending on the pathogenic organism and cell type. Evidence in recent years has also shown that activation of the Wnt/b-catenin pathway is able to reduce several inflammatory processes (Silva-García et al., 2014) that are triggered by an array of pathogens and proinflammatory stimuli, such as IFN-g, TNF-b and Nitric Oxide (NO), are able to increase the expression of Wnt/b-catenin signalling molecules indicating a connection between these pathways (Du et al., 2013; Gatica-Andrades et al., 2017). Furthermore, macrophages are able to use Wnt ligands to influence cell-fate decisions, such as cell death (Lobov et al., 2005).

Tumour-associated macrophages (TAMs) are a major part of the tumour microenvironment (TME) and they have been shown to play pivotal roles in the progression of a number of cancer types (Chanmee et al., 2014). Several studies have suggested that macrophage derived Wnt ligands are able to active the Wnt signalling pathway in tumour cells, however, it remains unknown whether Wnt ligands in the tumour are able to activate Wnt signalling in the TAMs (Yang et al., 2018; Zhan et al., 2017). Additionally, TAMs are known to express Wnt genes and are able to regulate the growth of tumour vasculature and potentially the tumours themselves (Newman and Hughes, 2012).

Concluding Remarks

As we continue to thrive in an era of immunotherapy and anti-cancertherapeutics, targeting the Wnt signalling pathway has become a thought-provoking focus for a number of research laboratories around the world. It is imperative to remember the importance and significance of Wnt during development and in normal cells, making manipulations of the pathway difficult. In recent years, huge leaps have been made in the development of antibodies (Vantictumab, OncoMed & Bayer) and decoy receptors (Ipafricept, OncoMed) able to modulate Wnt signalling in harmful, predominantly cancerous cells. However, several important questions remain regarding Wnt signalling and its effects in both healthy states and pathogenic or cancer settings.

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