**4. Host defense against** *Leishmania donovani* **infection in the context of Wnt5A signaling**

Macrophages, the primary sentinels of host immune response carry the potential to confront the challenge imposed by *L. donovani* infection. One important strategy adopted by macrophages to limit the pathogenicity from infection is production of ROS and nitric oxide, which are detrimental to the pathogen [57]. Interestingly the production of ROS and nitric oxide is often triggered by cytokine or chemokine signaling in the macrophages. IFN-γ is considered to be one of the major chemokines which bring about production of nitric oxide and ROS in macrophages [58]. IFN-γ null and IFN-γ receptor null mice carry enhanced infection load [50] substantiating the importance of IFN-γ in restraining infection. Overall, cytoskeletal actin modulations in association with organization of protein-lipid microdomains and transcriptional control of immune regulatory genes act in concert to antagonize the attempts of the parasite to settle into a favored niche within macrophages. These aspects of immune response to parasite infection are akin to the already known attributes of Wnt5A signaling as described previously in this chapter. Thus keeping in mind that *L. donovani* tries to subvert immune response by modulating lipid dynamics as well as cytoskeletal dynamics it is important to study the role of Wnt5A signaling in *L. donovani* infection.

Experimental evidence indicates that Wnt5A signaling and *L. donovani* infection are in mutual opposition. *In-vitro studies* have shown that the protein level of Wnt5A in *L. donovani* infected macrophages is significantly lower than that in the uninfected controls, with no significant change in Wnt5A mRNA level. The observed decrease in Wnt5A protein upon *L. donovani* infection is indicative of infection-induced suppression of Wnt5A signaling in the host macrophage. Protozoan parasites like *L. donovani,* are known to harbor a plethora of proteases (including metalloproteases) to counteract host immune response through cleavage of host proteins. GP63, one such well-studied metalloprotease, is expressed in significant amounts in both the promastigote and amastigote forms of the *L. donovani* parasite. Cleavage and destruction of host proteins such as AP1 by GP63 has already been reported [54]. Since, the reduction in Wnt5A protein upon infection of macrophages with *L. donovani* was inhibited by O-phenanthroline, a small molecule inhibitor of metalloproteases, it is possible that infection induced reduction in Wnt5A protein is brought about by the action of parasite specific metalloproteases like GP63. In view of the fact that Wnt5A signaling is known to boost immune homeostasis [40, 41], reduction in Wnt5A protein level may help the parasite to evade immune response. In contrary to our observation, the mRNA levels of Wnt5A are found to increase during mycobacterial and ehrlichial infection in macrophages [38, 59]. Changes in Wnt5A mRNA and protein levels may depend on the type and load of infection. Thus, further validation of proteomic data from various samples such as sera and spleen aspirates from *L. donovani* infected individuals will be needed to further analyze and understand the experimental findings.

**109**

*substrate.*

**Figure 2.**

*Wnt5A Signaling Antagonizes* Leishmania donovani *Infection*

of Wnt5A signaling in limiting *L. donovani* infection.

On the basis of the understanding that the steady state level of Wnt5A signaling is significantly reduced during *L. donovani* infection, we hypothesized that revamping Wnt5A signaling in macrophages might have a debilitating effect on parasite load. Indeed we found that upon treating macrophages with recombinant Wnt5A prior to infection there is significant reduction in parasite load. The decrease in the parasite load was seen to be dose and time dependent. Interestingly, a decrease in parasite load was also seen when Wnt5A was exogenously added to infected macrophages, suggesting that there may be a therapeutic role of Wnt5A signaling during *L. donovani* infection [42]. Depletion of Wnt5A signaling through application of IWP-2 or transfection with Frizzled5 (putative receptor to Wnt5A) siRNA resulted in enhancement of infection by *L. donovani*, corroborating the importance

There is evidence that Wnt5A signaling mediated killing of *L. donovani* within the macrophages is brought about by change in Wnt5A induced cytoskeletal and membrane dynamics. Revamping host Wnt5A signaling by exogenous Wnt5A leads to reduction in parasite survival probably because the lay out for a self-sustaining parasite niche in the form of parasitophorous vacuole (PV) is not compatible with the cytoskeletal alterations and associated endosomal/lysosomal vesicle movements induced by Wnt5A. Enhanced endolysosomal fusion in infected macrophages occurred through Wnt5A signaling in infected cells as judged by live cell microscopy

*Schematic representation of Wnt5A signaling-mediated inhibition of* Leishmania donovani *infection.*  Leishmania *promastigote interacts with the macrophage through different macrophage receptors. This interaction leads to the formation of parasite containing early endosome where several factors like low pH and increased temperature help in differentiation of the parasite into amastigote form. It secretes metalloproteases, which can degrade Wnt5A. Activated Wnt5A signaling modulates actin cytoskeleton and promotes phagolysosomal fusion leading to degradation of the parasite. Wnt5A signaling may also alter the expression of pro-inflammatory and anti-inflammatory cytokine genes thus promoting parasite clearance. PV, parasitophorous vacuole; ROCK , rho-associated coiled coil kinase; Rac, Ras-related C3 botulinum toxin* 

*DOI: http://dx.doi.org/10.5772/intechopen.87928*

*Wnt5A Signaling Antagonizes* Leishmania donovani *Infection DOI: http://dx.doi.org/10.5772/intechopen.87928*

*Vector-Borne Diseases - Recent Developments in Epidemiology and Control*

cytokine signature [37, 40, 41].

signaling in *L. donovani* infection.

**Wnt5A signaling**

*L. donovani* infection is accompanied by increase in anti-inflammatory cytokine expression, which may help the intracellular amastigotes to build a safe niche within the macrophage. Increase in anti-inflammatory cytokines is often associated with decrease in production of ROS or Nitric oxide, which is unfavorable for amastigote growth [54–56]. Host macrophage Wnt5A signaling may be instrumental in attenuating the effect of anti-inflammatory cytokines by maintaining a proinflammatory

**4. Host defense against** *Leishmania donovani* **infection in the context of** 

Macrophages, the primary sentinels of host immune response carry the potential to confront the challenge imposed by *L. donovani* infection. One important strategy adopted by macrophages to limit the pathogenicity from infection is production of ROS and nitric oxide, which are detrimental to the pathogen [57]. Interestingly the production of ROS and nitric oxide is often triggered by cytokine or chemokine signaling in the macrophages. IFN-γ is considered to be one of the major chemokines which bring about production of nitric oxide and ROS in macrophages [58]. IFN-γ null and IFN-γ receptor null mice carry enhanced infection load [50] substantiating the importance of IFN-γ in restraining infection. Overall, cytoskeletal actin modulations in association with organization of protein-lipid microdomains and transcriptional control of immune regulatory genes act in concert to antagonize the attempts of the parasite to settle into a favored niche within macrophages. These aspects of immune response to parasite infection are akin to the already known attributes of Wnt5A signaling as described previously in this chapter. Thus keeping in mind that *L. donovani* tries to subvert immune response by modulating lipid dynamics as well as cytoskeletal dynamics it is important to study the role of Wnt5A

Experimental evidence indicates that Wnt5A signaling and *L. donovani* infection are in mutual opposition. *In-vitro studies* have shown that the protein level of Wnt5A in *L. donovani* infected macrophages is significantly lower than that in the uninfected controls, with no significant change in Wnt5A mRNA level. The observed decrease in Wnt5A protein upon *L. donovani* infection is indicative of infection-induced suppression of Wnt5A signaling in the host macrophage. Protozoan parasites like *L. donovani,* are known to harbor a plethora of proteases (including metalloproteases) to counteract host immune response through cleavage of host proteins. GP63, one such well-studied metalloprotease, is expressed in significant amounts in both the promastigote and amastigote forms of the *L. donovani* parasite. Cleavage and destruction of host proteins such as AP1 by GP63 has already been reported [54]. Since, the reduction in Wnt5A protein upon infection of macrophages with *L. donovani* was inhibited by O-phenanthroline, a small molecule inhibitor of metalloproteases, it is possible that infection induced reduction in Wnt5A protein is brought about by the action of parasite specific metalloproteases like GP63. In view of the fact that Wnt5A signaling is known to boost immune homeostasis [40, 41], reduction in Wnt5A protein level may help the parasite to evade immune response. In contrary to our observation, the mRNA levels of Wnt5A are found to increase during mycobacterial and ehrlichial infection in macrophages [38, 59]. Changes in Wnt5A mRNA and protein levels may depend on the type and load of infection. Thus, further validation of proteomic data from various samples such as sera and spleen aspirates from *L. donovani* infected individuals will be needed to further analyze and understand the experi-

**108**

mental findings.

On the basis of the understanding that the steady state level of Wnt5A signaling is significantly reduced during *L. donovani* infection, we hypothesized that revamping Wnt5A signaling in macrophages might have a debilitating effect on parasite load. Indeed we found that upon treating macrophages with recombinant Wnt5A prior to infection there is significant reduction in parasite load. The decrease in the parasite load was seen to be dose and time dependent. Interestingly, a decrease in parasite load was also seen when Wnt5A was exogenously added to infected macrophages, suggesting that there may be a therapeutic role of Wnt5A signaling during *L. donovani* infection [42]. Depletion of Wnt5A signaling through application of IWP-2 or transfection with Frizzled5 (putative receptor to Wnt5A) siRNA resulted in enhancement of infection by *L. donovani*, corroborating the importance of Wnt5A signaling in limiting *L. donovani* infection.

There is evidence that Wnt5A signaling mediated killing of *L. donovani* within the macrophages is brought about by change in Wnt5A induced cytoskeletal and membrane dynamics. Revamping host Wnt5A signaling by exogenous Wnt5A leads to reduction in parasite survival probably because the lay out for a self-sustaining parasite niche in the form of parasitophorous vacuole (PV) is not compatible with the cytoskeletal alterations and associated endosomal/lysosomal vesicle movements induced by Wnt5A. Enhanced endolysosomal fusion in infected macrophages occurred through Wnt5A signaling in infected cells as judged by live cell microscopy

#### **Figure 2.**

*Schematic representation of Wnt5A signaling-mediated inhibition of* Leishmania donovani *infection.*  Leishmania *promastigote interacts with the macrophage through different macrophage receptors. This interaction leads to the formation of parasite containing early endosome where several factors like low pH and increased temperature help in differentiation of the parasite into amastigote form. It secretes metalloproteases, which can degrade Wnt5A. Activated Wnt5A signaling modulates actin cytoskeleton and promotes phagolysosomal fusion leading to degradation of the parasite. Wnt5A signaling may also alter the expression of pro-inflammatory and anti-inflammatory cytokine genes thus promoting parasite clearance. PV, parasitophorous vacuole; ROCK , rho-associated coiled coil kinase; Rac, Ras-related C3 botulinum toxin substrate.*

using live cell dyes [42]. Electron microscopic (EM) images showed Wnt5A induced increased membranous vesicle fusions with PV in the infected cells. The EM images also revealed a low abundance of the PV upon Wnt5A treatment. The apparent membranous wrappings in degraded PV, as suggested by EM may be due to the formation of autophagosomes through fusion of PV with membranous aggregates during cytoskeletal movements [42]. One of the strategies that a host may adopt through Wnt5A signaling is laminate the parasitophorous vacuole so that the solutes cannot easily reach the parasite thereby slowly starving the parasite to death. While laminating the parasitophorous vacuole it may also ensure that the NADPH oxidase is well assembled so as to generate adequate amounts of ROS, which could lead to killing of parasites. The membrane lamination on the parasitophorous vacuoles through enhanced cytoskeletal dynamics could also lead to increased PV-lysosomal fusion thereby promoting rapid degradation of the parasite. Our study demonstrates that Wnt5A signaling mediated killing of *L. donovani* in macrophages is abrogated when inhibitors of cytoskeletal proteins like Rac1 GTPase and Rho kinase are used, thus implying that the effects of Wnt5A signaling on infection are at least partly mediated through the small molecular weight actin associated GTPases. The possible mechanism of Wnt5A signaling mediated parasite clearance is depicted in **Figure 2**.

It will be important to validate the effect of Wnt5A signaling on *L. donovani* infection *in vivo* and also check the load of *L. donovani* infection in Wnt5A heterozygous mice (Wnt5A null are lethal). Analysis of the cytokine milieu *in vivo* upon activation of Wnt5A signaling at the onset of infection will also provide useful information about the mechanism of Wnt5A induced containment of infection.
