**3.1 Significance of phagocytosis**

Phagocytosis of pathogens is one of the most important features of the hostpathogen communications and interactions mediated by macrophages. This element of host defense by macrophages not only operates toward host protection at the onset of infection but also makes room for the initiation and amplification of intracellular signals that can potentially mature to the generation of antigen-specific T cell responses and creation of immunological memory (explained in **Figure 4**).

As described earlier in this chapter, Wnt5A signaling aids in maintaining a steady-state expression of CD14 and IFNβ, two of the many molecules involved in innate immune defense. Although it is not exactly clear how CD14 and IFNβ fit into the program of phagocytosis in exact molecular terms, it is documented that while CD14 is instrumental in the recognition of structural motifs like lipopolysaccharide (LPS) intrinsic to certain pathogens, both CD14 and IFNβ facilitate pathogen clearance through the initiation and propagation of macrophage TLR signaling during phagocytosis and activation of immune responses [10, 13] (**Figure 3**). Following pathogen engulfment and phagosome formation during phagocytosis, macrophages rely mostly on endosomal and lysosomal proteases and NADPH oxidase-generated reactive oxygen species for both pathogen clearance as well as processing and presentation of antigenic peptides to MHC molecules for presentation to T lymphocytes [31, 46] and translation to memory.

## **3.2 Need for Wnt5A signaling-assisted actin rearrangement/assembly**

At the core of all phagocytosis-related processes lies the involvement of the actin cytoskeleton through its influence on protein sorting/trafficking and intracellular organelle fusions that are crucial for the activation of phagosomal enzymes such as

#### **Figure 4.**

*A schematic of maturation of pathogen containing vesicle and its outcome: After phagocytosis of pathogen there is fusion of early endosome and endoplasmic reticulum (ER) with the phagosome which helps in the maturation of the phagosome and fusion with lysosome. This is important for both innate and adaptive immunity.*

NADPH oxidase and phagosome maturation [31, 47]. Several cytoskeletal GTPases such as Rac1 and Disheveled, lipid rafts, and actin-nucleating proteins such as Arp2/3 and formins partake of the cytoskeletal actin modulations that accompany macrophage phagocytosis and phagosome maturation [47–50]. There is evidence that Wnt5A signaling is important for such rearrangements of the actin cytoskeleton. Accordingly, Wnt5A signaling facilitates Rac1- Disheveled-lipid raft-dependent phagocytosis of bacteria and other foreign matter through modulations of the actin cytoskeleton [9]. Blockers of any of the cytoskeletal actin-associated signaling intermediates—Rac1, Disheveled, or lipid raft and cytochalasin-D, an inhibitor of actin assembly—are antagonistic to the effect of Wnt5A signaling on phagocytosis [9]. The influence of Wnt5A signaling on phagocytic uptake is usually dependent on the microbe under consideration, because while most bacterial species tested undergo facilitated phagocytic uptake by Wnt5A signaling in macrophages, phagocytic uptake of *Leishmania donovani* remains unaffected by it [11]. Perhaps Wnt5A-facilitated internalization encompasses distinct membranous domains depending on the availability of cognate receptors, which are not equally compatible with all microbes. That Wnt5A signaling also facilitates phagosome-lysosome fusion during phagosome maturation which is evident from the augmented appearance of lysosomal markers such as cathepsins in Wnt5A-induced phagosomes of bacteria-infected macrophages [12]. Wnt5A-facilitated alteration in cytoskeletal actin assembly that correlates with phagosome-lysosome fusion is concomitant with the killing of several microbes including bacterial pathogens (*Pseudomonas aeruginosa*, *Streptococcus pneumoniae*, etc.) and even *Leishmania donovani*, although it gets internalized independent of Wnt5A signaling [11, 12]. The mechanism of microbial killing is discussed at greater length in the following section of this chapter. Microbial killing is furthermore facilitated by Wnt5A-responsive NADPH oxidase activity, which is associated with cytoskeletal actin-dependent assembly of NADPH oxidase subunits [11]. Interestingly, nonpathogenic laboratory strains of bacteria that are engulfed by macrophages in increased numbers by Wnt5A signaling are not necessarily killed by it like the pathogenic bacterial strains [9, 12]. Such discrepancy in the fate of internalized microbes may be an outcome of notable differences in the interaction of different microbial components with Wnt5A-regulated cytoskeletal actin rearrangements. The interrelation between Wnt5A signaling and Ehrlichia infection is especially noteworthy in this context [51].

In light of the fact that the cytoskeletal actin-assisted phagosome is the originator and communicator of many signals generated by phagocytozed cargorecognizing molecules such as TLR, NOD1, and NOD2 [35, 52, 53] (**Figure 4**), it is quite likely that the consequences of Wnt5A-assisted phagocytosis are numerous. Association of Wnt5A signaling with TLRs has already been reported [54]. Careful analysis of the consequences of such associations is important.

#### **3.3 Role played by other Wnts and costimulatory molecules of Wnt signaling**

Wnts other than Wnt5A are known to regulate macrophage phagocytosis as well. For example, the Drosophila Wnt has been reported to stimulate phagocytic uptake in the S2 cell, a macrophage-like line [55]. Moreover, Wnt1, Wnt7A, and Wnt3A have been reported as phagocytic modulators [56, 57]. The association or relation of these different modes of phagocytosis with Wnt5A signaling and cytoskeletal actin rearrangements is yet to be explored. At this point of our understanding of Wnt signaling with respect to phagocytosis, regulatory roles played by LRP5/6 and ROR, which act as co-receptors to Wnts [22, 58], remain unclear. It also remains to be seen if the influence of Wnt5A signaling on phagocytosis is in the canonical or noncanonical mode or is in fact an intermediary between the two depending on the context of infection, the available receptors, and coactivators.

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

## **4. Wnt signaling-induced actin-dependent autophagy-assisted xenophagy by macrophages and the potential link with antigen processing/presentation**

#### **4.1 Autophagy-assisted xenophagy**

Several pathogenic microorganisms try to adapt to the intracellular milieu of macrophage creating a niche for their survival [59–61]. Nevertheless, as described earlier in this chapter, the host macrophage tries maneuvering elimination of infection by pathogens by several means. It has been reported that following phagocytosis of microbes by macrophages, the host autophagy machinery comes into play in the ultimate event of clearance of bacteria and other engulfed microbes (xenophagy) through coordinated alterations of the actin cytoskeleton. Autophagy involves the turnover and clearance of damaged organelles and proteins by the cell under both normal conditions as well as under stress in the maintenance of cellular homeostasis [62, 63]. During infection with pathogens, the autophagy program is often utilized for the incapacitation and eradication of engulfed pathogens [26, 64].

#### **4.2 Role of Wnt signaling and cytoskeletal actin in autophagy-assisted xenophagy**

Wnt signaling has been reported to play a significant role in the autophagyassisted xenophagy of engulfed microbes by macrophages. Wnt5A signaling, for instance, has been documented to be an integral component of this theme in the killing of several bacterial pathogens through utilization of a Rac1-Disheveled-actin cytoskeleton circuit that involves interactions among several autophagy-associated proteins like microtubule-associated protein 1B-light chain 3B (LC3B), autophagyrelated 5 (ATG5), ATG7, and Unc-51-like autophagy-activating kinase 1 (ULK1) [12]. The different nuances of Wnt5A signaling in connection with the actin cytoskeleton are depicted in **Figure 5**. Pathogen killing through autophagy machinery is blocked with the use of cytochalasin-D, an inhibitor of actin assembly as well as with inhibitors to Rac1 and Disheveled [12]. Although Wnt5A-assisted killing of *L. donovani* in macrophages has not been shown to directly involve autophagy, electron micrographs of *L. donovani* harboring parasitophorous vacuoles, which display distinct membranous aggregates, suggest that *L. donovani* containing parasitophorous vacuoles may be subjected to lysis by the host autophagy circuit activated by Wnt5A signaling [11]. The inactivation or lysis of microbe-carrying vacuoles, which happens in due course through fusion of autophagy-destined phagosome or autophagosome with the lysosome, may also be facilitated by Wnt5A signaling [12]. Although cholesterol and other lipids are known to partake of both Wnt5A signaling and actin dynamics [65, 66], at this stage much remains unknown about the specific roles of cholesterol and other lipids in the process of actin modulation during phagocytosis and autophagic clearance of bacteria and other microbes. It also remains to be seen if Wnt5A signaling during autophagy belongs strictly to the noncanonical mode or canonical mode based on the involvement of β-catenin.

#### **4.3 Potential link with antigen presentation/adaptive immunity**

In view of the fact that the autophagic or rather xenophagic removal of pathogens by macrophages involves reorganization and fusion of intracellular vesicles associated with at least partial lysis of pathogens, the processing and presentation of pathogen antigens to MHC molecules are a likely event during xenophagy in infected macrophages [67, 68]. Thus, autophagosome formation, autophagosome lysosome fusion, and T cell activation by the presentation of processed pathogenic

**Figure 5.**

*Schematic of Wnt5A signaling aided bacterial killing: Both autocrine and paracrine modes of Wnt5A signaling can lead to increase in phagocytosis of pathogenic bacteria. After phagocytosis, the Wnt5A mediated cytoskeletal modulation leads to fusion of early endosome and lysosome with the pathogen containing phagosome. Wnt5A signaling also activates Rac1 and Unc like kinase 1 (Ulk1) for initiation of autophagy. The subsequent steps of maturation lead to killing of pathogen in an autophagy dependent process (xenophagy).*

antigens may prevail as a continuum during immune defense depending on the nature and degree of the infection. Given the intrinsic association of Wnt signaling with cytoskeletal dynamics and autophagy [11, 12], it is quite likely that Wnt signaling will influence the antigen processing and presentation linked with autophagy in infected macrophages. Detailed investigation in this respect, although important, remains to be documented.

#### **5. Concluding remarks**

Given the important role played by Wnt ligands in the transmission of signals associated with cytoskeletal modulation and transcriptional regulation which are part and parcel of host-pathogen communications [27–29, 69], a combination of Wnt signal transduction cascades is expected to hold a fundamental standing in the immune defense program operated by macrophages in both innate and adaptive immunity. Phagocytosis, autophagy/xenophagy (intracellular microbial killing), and a steady-state expression of immune defense molecules through transcriptional regulation appear as some of the major players of the immune defense program operated by Wnt signaling.

In respect of transcriptional regulation of immune defense molecules by steadystate Wnt5A-signaling as described in this chapter [10], it is not understood exactly what dictates the nuclear translocation of p65 and not the other NFκB isoforms for specific modes of gene expression. Additionally, how this regulation fits in with the activity of other major transcription factors like NFAT and AP1 in the macrophage is also not clearly understood. Moreover, details of the context dependence of

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

Wnt5A signaling, wherein a certain level and mode of signal transmission will be beneficial for immune response, but excess will cause inflammation and disorder [70–72], remain largely unclear. Besides, a clear concept of how actin cytoskeletonassociated proteins such as Rac1 promote both NFκB activity as well as cytoskeletal rearrangements for phagocytosis and autophagy is yet to be achieved [10, 12]. Whether nuclear translocation of NFκB is a natural function of actin assembly or is executed by a separate pool of Rac1 associated cytoskeletal proteins is an important matter that deserves investigation.

With regard to phagocytosis and autophagy-assisted xenophagy, the molecular details of the actin rearrangements with actin binding proteins and the processing and presentation of antigens remain to be deciphered. This brings into question how different host-pathogen interactions within macrophages are guided by modulations of the actin cytoskeleton. Of special interest in this context is the interaction of the actin cytoskeleton with pathogenic mycobacteria, which thrive in self-generated niches within macrophages [60, 73]. The interrelation between different modes of Wnt signaling and mycobacterial infection, although much studied [74, 75], needs to be better understood with respect to actin dynamics. Now that Wnt5A signaling has been shown to play a major role in the regulation of actin cytoskeletal modulation and autophagy [11, 12, 76], future experiments addressing whether this can also facilitate the adaptive immune response through antigen processing and presentation may prove fruitful.

At this juncture of our understanding of Wnt signaling and immune response by macrophages, it is important to know how the different Wnt ligands operate in the regulation of immune response by the different types of macrophages that are distributed in different tissues under the varied conditions of intracellular milieu and infection. Macrophages (microglia) present in the brain and spinal cord maintain an active immune defense scheme against pathogens that affect the central nervous system. Alveolar and airway macrophages likewise protect the respiratory tract and lungs from the toxic effect of infectious agents. Peritoneal macrophages of the peritoneum and Kupffer cells of the liver also encounter and confront infectious agents for host protection. Quite naturally, the roles played by Wnt signaling in the combat mechanism of each macrophage type in its paradigm of immune defense is expected to be different at least to some extent on account of potential variations in cellular environmental cues and modes of host-pathogen interactions.
