**2. Sustenance of immune defense by macrophages through a steady state of transcriptional activation by Wnt signaling**

### **2.1 Significance of constitutive transcriptional activation in macrophages by Wnt signaling**

Macrophages have long been acknowledged for executing immune defense against microbial pathogens through diverse means of signaling that include several transcription factors including NFκB, AP1, and NFAT [27–30]. The ability of macrophages to recognize and engulf pathogens, deliberate NADPH oxidase activity, and process antigens for presentation to MHC molecules and T cell activation place macrophages quite aptly at the crossroad of innate and adaptive immune defense programs [31–33]. Surely, macrophages have in-built mechanisms to execute innate immunity and translate it to adaptive immune response. However, not much is known about the molecular details of how macrophages are naturally geared to operate in such innate and adaptive modes of immune defense. We recently demonstrated that NF-κB (p65) [34], a transcription factor functioning at the core of our immune system, remains activated at a basal level in macrophages through a steady state of Wnt5A signaling. Administration of inhibitor of Wnt production2 (IWP2) to macrophages in culture or depletion of Wnt5A or Frizzled5 (putative Wnt5A receptor) gene expression in macrophages by silencing gene transcription through small interfering RNA blocks constitutive p65 activation and the steady-state immune activity of macrophages [10]. Sustained presence of the Wnt5A-p65 axis can potentially bridge innate and adaptive immune responses through regulation of the expression of immune response genes, such as CD14, interferons (IFN)s, and MHC, and elaboration of immune signaling networks that involve major immune response molecules such as the Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-containing proteins (NOD) during challenge by pathogens [13, 35, 36]. The interrelation of this basal level Wnt5A-p65 signaling with other major transcription factors and coactivators of Wnt signaling that mediate immune response by macrophages remains to be deciphered at the molecular level.

#### **2.2 NF-κB transcription factors**

NF-κB transcription factors comprise a family of five members: p52, p50, p65 (RelA), c-Rel, and RelB, which regulate gene transcription as combinatorial dimers [34, 37, 38]. These dimers remain or become activated through different modes depending on the physiological context of cell signaling. In the classical mode of activation, the homo and heterodimers are translocated to the nucleus for gene expression after being released from the IκB-bound states in the cytoplasm in response to different stimuli that lead to proteasome-assisted IκB degradation through activation of the IκB kinase IKK2/β [34]. The p65 homo and heterodimers while being responsible for inflammatory gene expression are also significantly involved in the sustenance of innate immune response gene expression in a context-dependent manner [10]. Some of the NF-κB (p65) responsive immune response genes include CD14, MHC, and IFNs. A schematic of NF-κB activation is shown in **Figure 2**.

#### **2.3 Wnt5A signaling-mediated activation of transcription**

As mentioned earlier in this chapter, Wnt5A is one of several members of the large family of Wnt glycoprotein ligands. Frizzled-5, Frizzled-4, and ROR1 are putative receptors for Wnt5A. It is to be noted that although modified versions of selective Wnt-Frizzled complex structures have been solved [39], none of the ligand-receptor

*Wnt Signaling Regulates Macrophage Mediated Immune Response to Pathogens DOI: http://dx.doi.org/10.5772/intechopen.86433*

#### **Figure 2.**

*An overview of NFKB activation pathway in the macrophage: During steady state a basal level of stimulus by Wnt signaling keeps IKK enough activated to result in inactivation of IκB and translocation of a certain pool of NFκB transcription factor (p65 homodimer) to the nucleus. A minimum pool of transcription factors contributes to survival and vigilance for immune response. In the activated state, during inflammation and chronic infection, stimuli (TNFα, LPS, IL1β) lead to an increase in NFκB combinatorial dimers in the nucleus.*

complexes have been truly biochemically characterized in their physiological contexts. In the noncanonical mode of Wnt signaling of which Wnt5A is a representative, Wnt5A-Frizzled-ROR or Wnt5A-Frizzled-initiated signaling alters the activity of Rho/Rac family GTPases through differential activation of Disheveled [10, 40]. Within the Frizzled family of cell surface receptors, Frizzled2, Frizzled5, and Frizzled4 are some of the putative receptors for Wnt5A [17, 41, 42]. It is not known if Disheveled activation by Wnt5A signaling acts in concert with or is regulated by heterotrimeric G proteins, given that Frizzled receptors are homologous to heterotrimeric G protein-coupled receptors. The involvement of β-catenin by Wnt5A signaling is governed by the availability of receptors and cytoplasmic signaling intermediates [20, 43]. The subsequent activation of transcription factors such as AP1, NFAT, and NF-κB through complex signaling networks and crosstalk, either dependent or independent of nuclear translocation of β-catenin (explained in **Figures 1** and **2**), could lead to elaboration of context-dependent immune responses (**Figure 3**).

The basal Wnt5A-Frizzled5 signaling-dependent NF-κB (p65) activity in macrophages that we observed is at least partly accountable for the steady-state expression of CD14/IFNβ, the promoter sequence of which at the genome level contains p65 binding elements [10, 13] (**Figure 3**). The constitutive p65 activity in the nucleus also contributes to sustaining Wnt5A expression [10]. Accordingly, the self-sustaining Wnt5A-p65 axis responsive CD14 and IFNβ expression helps to initiate and coordinate several aspects of macrophage function including interaction of pathogen recognition with TLR signaling, thus enabling adaptation to protective immune responses to bacteria, bacterial LPS (lipopolysaccharide), and virus as explained in **Figure 3**. The Wnt5A-NF-κB (p65) responsive gene expression declines upon

#### **Figure 3.**

*A schematic of Wnt5A-p65 axis: Wnt5A binds with it's putative receptor Frizzled5 (FZ) and transmits signal through intermediates like Disheveled (Dvl), trimeric G-proteins (Gα, β, ƴ ) activating Rac1. Activated Rac1 helps in translocation of NFκB from cytosol to nucleus via activation of IKK and proteasomal degradation of IKK-phosphorylated IκB. The translocated p65 in the nucleus helps to maintain expression of proteins such as CD14, IFNƴ, IFNb, MHC, needed for pathogen detection and clearance, and Bcl2, needed for cell survival. Amplification of signals by CD14-assisted molecules such as TLRs facilitate pathogen recognition and clearance.*

exposing macrophages to an IKK2-specific inhibitor [10]. Wnt5A signaling is also responsible for a basal level of secretion of IFN-γ, another important regulator of innate immune signaling in macrophages. The steady-state Wnt5A signaling and NF-κB activity also promote macrophage survival through the expression of NF-κBresponsive survival genes such as Bcl2 [10]. These data are consistent with the dearth of survival of NF-κB-deficient mice due to different kinds of infection and apoptotic cell death [44]. The Wnt5A-Frizzled5 signaling-assisted constitutive p65 activity is dependent on Rac1 activation, which lies upstream of IKK2 activity [10]. The detailed mechanism of how the Rac1 GTPase activates IKK in a Wnt5A signaling-dependent mode is yet to be explored. It also remains to be tested how Wnt5A-responsive innate immune functions in macrophages relating to pathogen recognition and activation of several intracellular signaling pathways translate to adaptive immune responses encompassing antigen processing/presentation and lymphocyte activation.

#### **2.4 Signaling and transcriptional activation by other Wnts**

In light of the fact that Wnts comprise a large family of glycoprotein ligands sharing considerable amino acid sequence homology and bind to cell surface receptors that are equally homologous [21], the schemes of regulation and sustenance of immune responses in macrophages by Wnt signaling are likely to be manifold. Several reports have outlined the importance of canonical Wnt signaling and β-catenin in the development, sustenance, and elaboration of memory and effector T cells that comprise a crucially important component of immunity to infectious pathogens [45]. The role of the TCF family of transcription factors in this respect has generated considerable interest in our understanding of the importance of Wnt signaling in immune homeostasis. However, the precise role of canonical Wnt signaling by β-catenin and TCF transcription factors in macrophages in the generation and sustenance of T cell-mediated immunity remains unclear.
