The Tale of Mastering Macrophage Environment through the Control of Inflammasome-Mediated Macrophage Activation and cAMP Homeostasis by the Protozoan Parasite *Leishmania*

*Anindita Bhattacharjee, Arunima Biswas and Pijush K. Das*

#### **Abstract**

*Leishmania,* being an intelligent protozoan parasite, modulates the defensive arsenals of the host to create a favorable niche for their survival. When the intracellular parasite is encountered by the host, multimeric complexes of inflammasomes get assembled and activated, thereby leading to genesis of inflammatory response. In order to subvert host defensive strategies, *Leishmania* utilizes their cyclic adenosine monophosphate (cAMP) and cAMP-induced response to neutralize macrophage oxidative damage. In this chapter, we summarize our current understanding of the mechanisms of inflammasome activation in macrophages and cAMP homeostasis of the parasite, leading to parasite viability within the macrophages and establishment of infection. Furthermore, we took into account, recent progresses in translating these research areas into therapeutic strategies, aimed at combating macrophage associated diseases.

**Keywords:** inflammasome, NLRP3, cAMP homeostasis, phagosome, *Leishmania*, macrophage

#### **1. Introduction**

Leishmaniases involve a broad spectrum of neglected tropical diseases that are caused by kinetoplastid parasites belonging to the genus *Leishmania*, that is transmitted by female sandflies belonging to the genus *Phlebotomus* [1]. From the clinical manifestations and symptoms occurring in hosts harboring various species of *Leishmania,* three major forms of *Leishmania* can be delineated: visceral, cutaneous and mucocutaneous leishmaniasis. Visceral leishmaniasis is the chronic and often fatal, if left untreated, form of the disease primarily caused by *Leishmania donovani*

and *Leishmania infantum,* infecting visceral organs including bone marrow, liver and spleen. Cutaneous leishmaniasis, generally caused by *Leishmania major*, produces self-healing ulcerative lesions on the skin. Another variant of cutaneous leishmaniasis is mucocutaneous leishmaniasis that is characterized by damaged oro-nasopharyngeal tissues. The infective stage, i.e., the metacyclic promastigotes, are found to be embedded in a proteophosphoglycan-rich gel derived from the parasite itself in the anterior mid-gut of the sandfly, which plays important role in parasite regurgitation during blood meal feeding of the sandfly [1]. The moment the parasites enter the host system, they are rapidly phagocytosed by the macrophages where they differentiate into aflagellar, nonmotile amastigotes that multiply within the acidic phagolysosomal compartment of the macrophages [2]. In order to survive within the unfriendly environment of the macrophage, *Leishmania* evolved several approaches to counter the microbicidal power of the macrophage and to mount an effective immune response against the parasite. After the primary encounter of the parasites with neutrophils during early stages of host infection, the monocytes and macrophages in the blood stream participate in the act of engulfing *Leishmania* parasites by phagocytosis. Monocytes that express CD11-c on their surface have recently been identified as the major cellular micro-environment for *Leishmania* to proliferate and also acts as a reservoir of *Leishmania*, providing ample number of parasites required for infecting neighboring cells [2]. On the other hand, recruitment and activation of the inflammatory monocytes protect the host system by limiting proliferation of the parasites by reactive oxygen species (ROS) and inducible nitric oxide synthase (iNOS) generation. However, tissue macrophages become the primary host cells responsible for parasite elimination after infection has been established in the tissues. The macrophages are activated by two components of the immune system like interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF- α), that synergistically induce iNOS expression, the enzyme responsible for production of free radical NO which actively kills *Leishmania* parasites through oxidative stress induction. Apart from the induction of oxidative stress by the macrophage, monocyte-derived macrophages were found to be responsible for activation of inflammasomes and secretion of cytokines like Interleukin-1 β (IL-1β) upon parasitic infection [3]. Inflammasomes are cytosolic complexes that gather upon sensing microbial molecules or cellular stress, leading to cytokine processing and an inflammatory programmed cell death called pyroptosis, activated by caspase-1. Different species of *Leishmania*, when subjected to analysis against inflammasome activation, were found to be capable of inducing the macrophages to release IL-β by activation of NLRP3 (Nod-like receptor), CASP1 (Caspase-1) and ASC (Apoptosis-associated speck-like protein containing CARD) [4]. IL-1β production by activated NLRP3 inflammasome induces specific signaling pathways triggering NO synthesis leading to parasite killing and disease resistance. In a recent study, it is documented that *Leishmania* lipophosphoglycan (LPG) activates Caspase-11 which in turn activates the non-canonical NLRP3 inflammasomes. Strikingly, the amastigote form of the parasite causes lesser activation of inflammasomes owing to the fact that they display fewer LPG on their surface as compared to the promastigote forms. Moreover, the amastigotes destruct the NLRP3 inflammasome activation pathway in the macrophages by targeting histone H3 [5]. Therefore, it can be considered as an excellent strategy adapted by the parasites to combat the hostility offered by the macrophages against them.

Another major modulator of cytological events in *Leishmania* and other related kinetoplastids is cAMP (cyclic adenosine monophosphate), which is one of the primary factors responsible for parasite transformation and parasite survival. cAMP *The Tale of Mastering Macrophage Environment through the Control… DOI: http://dx.doi.org/10.5772/intechopen.102671*

plays a major role in differentiation-induced cell cycle arrest in G0/G1 phase in both *Leishmania* and *Trypanosoma* [6]. It has significant role in modulating promastigote proliferation, flagellar length and motility and regulation of mitochondrial membrane potential [7]. Moreover, the oxidative burst that the parasites encounter as the first line of defense in mammalian macrophage is associated with elevation of cAMP in the parasite triggering cell cycle arrest and stage differentiation [8]. Thus, it is quite evident that there is a delicate interconnection between the immune response offered by the macrophage upon *Leishmania* infection and the cAMP homeostasis inside the parasite that prevents their killing withing the host. This chapter will discuss the host–parasite interaction, inflammasome activation by the infected macrophages and cAMP homeostasis within the parasite leading to parasite survival.

#### **2. Epidemiology of leishmaniasis**

Visceral leishmaniasis, also known as kala-azar, is a deadly disease and in over 95% of instances it is found to be direful when left untreated. Approximately 50,000–90,000 new incidents of visceral leishmaniasis seem to occur globally each year in countries like Bangladesh, Brazil, China, Ethiopia, India, Kenya, Nepal, Somalia, South Sudan and Sudan. Cutaneous and mucocutaneous leishmaniasis occur in South American countries, Mediterranean river basin, the Central Asia and Middle East. Over 90% of mucocutaneous leishmaniasis cases occur in Bolivia, Brazil, Ethiopia and Peru [9]. The diseases collectively affect more than 1 million people every year, visceral leishmaniasis being the main reason for the death of a vast population of about 20,000–40,000 people annually [10]. Apart from humans, the primary host of the diseases, dogs, rodents, other mammals and few reptilian species act as the reservoir hosts of leishmaniases [11].

#### **3. Parasite infection and activation of parasite-dependent host biology modulation**

#### **3.1 Initial events of parasite infection and macrophage activation**

The disease manifestation of VL strictly relies on host immunocompetency ranging from asymptomatic forms to severe disease conditions, which if left untreated, can be fatal. Clinical conditions such as HIV infection or immunosuppression due to any drug treatment leading to immunodepression in host weaken the efficiency of the host immune system to deal with the infection and permit recurrence of the disease. The contrast in host–parasite interactions between cutaneous and visceral leishmaniasis is quite noteworthy, suggesting the role of infecting species of *Leishmania* in diversification of complexity of immune responses to the pathogen [12]. Resistance and susceptibility of the host to the infection is associated with various genetic factors impacting both severity of the disease and its diagnosis in case of visceral leishmaniasis. One of the most crucial factors that determine the clinical outcome of the disease is host immune response upon parasite entry. One of the components of innate immunity, the complement system, is activated immediately after penetration of the dermis by the promastigotes, resulting in effective clearance of about 90% of all injected parasites within a moment or two [13, 14]. Cells of the innate immune system including dendritic cells, NK (natural killer) cells, T cells and the cytokines they release,

altogether participate to mount immune response against *Leishmania*. IL-12 released by the dendritic cells activates NK cells that produce IFN-γ that enhance killing mechanisms in macrophages, the main target of *Leishmania* [15]. Apart from interleukins and interferons, tumor necrosis factors (TNFs) play a pivotal role in both parasite clearance and tissue damage in liver and spleen, respectively. *Leishmania* parasites modify the chemokine profiles of the host–parasite persistence is promoted. IFN-γ release triggered by Th1 response correlates with infection resistance by induction of some leishmanicidal mechanism in the phagocytes. However, Th2 response results in infection susceptibility and disease manifestation due to replication of intracellular parasite as macrophage activation is inhibited [16]. Thus, symptoms and extent of disease manifestation largely depends on immunocompetency of the host and the fine interplay between the pathogen and the immunological and genetic characteristics of the infected host.

#### *3.1.1 Recognition and uptake*

During the parasite's transfer from its vector to the vertebrate host, neutrophils and macrophages are quickly recruited to the sand fly bite site [17]. The parasites secret proteophosphoglycans inside sandfly midgut which acts as a potent stimulator for recruiting macrophages at the site of infection as found in both *L. mexicana* and *L. infantum* [18, 19]. Parasites initially infect the neutrophils but since neutrophils are unsuitable for parasitic differentiation into the amastigote forms, they start infecting the macrophages, their actual site of transformation [20–22]. As a result, the macrophage is a crucial host cell for parasite infection and persistence. The parasite flagellum initiates the contact between the promastigote and the macrophage. This may induce the parasite to release intrinsic survival factors, hence modulating phagocytic activity of the macrophages [20]. The complexity of *Leishmania*-macrophage interaction largely depends on the type of macrophage receptors involved with parasite recognition. Complement receptor-mediated uptake of the parasite results in parasite survival within the macrophage phagosomes because of inhibition of oxidative burst and inflammation, and lysosomal markers like Cathepsin D and LAMP1 accumulation. On the other hand, increased inflammatory conditions developed by fibronectin receptor-mediated uptake leads to parasite clearance. Other receptors including mannose receptors and FCγ receptors trigger inflammatory responses and NADPH oxidase activation in the phagosomes, respectively [23].

After the event of parasite recognition by macrophage cell surface, promastigotes are internalized by cholesterol-rich caveolae for both *L. chagasi* and *L. donovani* uptake [24–26]. However, lipid microdomains is found to have no effect on amastigote phagocytosis, highlighting the importance of membrane lipid-microdomains in the phagocytosis of *Leishmania* by the mammalian macrophages [24]. It is demonstrated in recent studies that lipid microdomain synthesis is promoted by *Leishmania* promastigotes by conversion of sphingomyelin into ceramide, the main constituent of lipid microdomain, by the activation of host acid sphingomyelinase [27]. *De novo* ceramide production is induced by the parasite at late stage of infection leading to disruption of lipid microdomain and impaired antigen presentation [27]. Moreover, *L. donovani* targets a macrophage transcription factor, SREBP2, which promotes parasite internalization and persistence through regulation of macrophage membrane cholesterol and mitochondrial ROS generation [28]. As a result, precise modulation of lipid microdomains is likely to be one of the important actors in host–parasite interactions in *Leishmania* infection.

*The Tale of Mastering Macrophage Environment through the Control… DOI: http://dx.doi.org/10.5772/intechopen.102671*

#### *3.1.2 Phagosome maturation and parasite differentiation*

Following phagocytosis of the promastigotes by the macrophages and their internalization into the phagosome, the parasites fuse with lysosomes and adapt to the hostile environment where they must survive for disease manifestations. Despite the fact that this is one of the most difficult habitats for most infectious pathogens, *Leishmania* is one of the few protozoan parasites that can survive and reproduce in such conditions. Phagosomes that contain *L. donovani* promastigotes showed reduced fusogenicity towards lysosomes and late endosomes [29, 30]. LAMP1, a lysosome marker, was employed to the parasitophorous vacuoles leading to reorientation of the parasites with their cell body facing the macrophage nucleus and the flagellum in the direction of the periphery of the cell. This orientation of the parasites endorsed the parasitophorous vacuole to move outwards causing cell injury, accelerated lysosome docking followed by exocytosis. Some of the parasite containing lysosomes might fuse with the parasitophorous vacuoles promoting transformation of promastigote into amastigote, whereas cell injury might disrupt the integrity of the plasma membrane and the capacity of the host to fight infection [31].

Differentiation from promastigote to amastigote is triggered by an increase in temperature from 22–37°C and a decrease in pH from 7.2 to 5.5 in mammalian phagolysosomes. Furthermore, iron uptake followed by hydrogen peroxide generation has been found to be a significant trigger for parasitic differentiation in *L. amanzonensis* [32, 33]. *Leishmania* iron transporter (LIT1) triggers the conversion of ROS into hydrogen peroxide by the enzyme iron superoxide dismutase; hydrogen peroxide being the major trigger for promastigote-to-amastigote differentiation to occur [34]. From previous studies, it was well documented that there is a close association between this transformation and the elevated levels of cAMP and cAMP-dependent Protein Kinase A (PKA) within the parasite [35]. cAMP plays a very crucial role in the transformation and regulation of the cell cycle of the parasites. cAMP acts as a cyto-protector which increase peroxide neutralizing capacity of the parasite increasing their chance of survival and is also known to trigger G1 arrest [8]. Several isoforms of leishmanial phosphodiesterases (LdPDEA and LdPDED) also showed roles in controlling the cAMP levels during transformation along with the leishmanial receptor adenylate cyclases (LdracA and LdracB) and pyrophosphatases (V-H+ PPase) proving beyond doubt the importance of cAMP signaling cascade in the parasite [35, 36].

#### *3.1.3 Macrophage activation: host: parasite interaction*

Macrophages, apart from acting as a phagocytic cell, respond to and regulate different signaling molecules [37]. Circulating monocytes are the precursors of tissue macrophages that secrete various antimicrobial and immunoregulatory molecules capable of inactivating pathogens through ROS and NO generation [38–40]. Monocyte–macrophage lineage show notable plasticity and can modify their physiology according to the environmental stimuli giving rise to diversified cell population with different functions [41, 42]. The state of activation of macrophages can be changed in response to different cytokines, growth factors and microbial molecules. When stimulated by TNF-α or IFN-γ or lipopolysaccharide, macrophages undergo classical activation that is characterized by surface marker CD80 expression [43, 44]. On the other hand, macrophages undergoing alternative activation is induced by IL-4 and IL-13 by the activation of a common receptor, IL-4R [45]. Production of high levels IL-13, CCL14, CCL17, CCL18, CCL22, IL-10, TGF-β, urea, and ornithine which

is an essential substrate for both polyamine and collagen synthesis, are observed in alternatively activated macrophages [44, 46, 47]. One of the mechanisms for the establishment of intra-macrophage parasite infection is the inhibition of host defense mechanism which is achieved by inhibiting inflammatory cytokine secretion and apoptosis. One such target of *L. donovani* promastigotes is AKT signaling and its downstream components, β-catenin and FOXO-1, modulation of which inhibits both cytokine production and apoptosis [48]. Thus, the initial interaction of *Leishmania* with macrophages leads to their activation and polarization that is essential for the survival of the parasite inside the macrophage and establishment of disease manifestation.

#### **3.2 Inflammasome activation by** *Leishmania***: mastering the macrophage environment exploiting macrophage biology**

Upon detection of pathogenic organisms, the cytoplasm of the cells of innate immunity assembles multiprotein complexes called inflammasomes that cause an inflammatory programmed cell death called pyroptosis. The nucleotide-binding domain leucine-rich repeat protein (NLR) family is the widely studied inflammasome that is activated by cell membrane damage-inducing pathogens and molecules (**Figure 1**) [49]. Caspase-1 activation is promoted upon NLRP3 activation and oligomerization which leads to ASC polymerization exposing the CARD domains of the ASC, leading to recruitment of Caspase-1 through CARD/CARD interaction. The NLRP3 inflammasome undergoes both canonical and non-canonical activation. Initially, TLR (toll-like receptors) or TNFRs are stimulated by microbial components or TNF-α leading to numerous inflammatory gene transcription, Nlrp3, Casp11 and Il1b for instance. But inspite of the presence of a first signal, a second signal is required for canonical NLRP3 activation that occurs via pore formation by the microbial toxins, and subsequent rupture of the host cell membrane, resulting in K<sup>+</sup> efflux

#### **Figure 1.**

*Interaction of* Leishmania *promastigotes with macrophage receptors triggers phagocytosis of the parasite and release of intrinsic survival factors by the pathogen that modify phagosome synthesis and results in inhibition of proinflammatory pathways. The schematic diagram is the summary of the fine interplay between inflammasomeinduced and cAMP-mediated differentiation of the parasite through modulation of ROS and activation by Ld receptor adenylate cyclases (LdRacs) by acidocalcisomal pyrophosphatase (V-H+ PPase) resulting in upregulation of cAMP, respectively. PPi = inorganic pyrophosphate pool, AC = acidocalcisome.*

#### *The Tale of Mastering Macrophage Environment through the Control… DOI: http://dx.doi.org/10.5772/intechopen.102671*

and decrease in K<sup>+</sup> concentration the cytoplasm [50]. Apart from potassium efflux, lysosomal cathepsin and ROS production is also essential for canonical activation of NLRP3. In contrast, non-canonical activation of NLRP3 inflammasomes is promoted by Caspase-11 which is activated by bacterial LPS. Different species of *Leishmania* use different strategies of limiting or inhibiting inflammasome activation in the macrophages. *L. mexicana* and *L. major*, as demonstrated by Shio et al., use GP63, a virulence factor that inhibit IL1b production in THP-1 cells, either by ROS inhibition or by inflammasome components cleavage [51]. Inhibition of NLRP3 inflammasome by *L. donovani* is achieved by A20, a negative regulator of NF-κβ and UCP2, mitochondrial uncoupling protein 2 manipulation by inhibiting ROS generation [52]. Leishmania infection induces the expression of UCP2 leading to downregulation of ROS generation by the macrophage, thus probably averting ROS-mediated inactivation of protein tyrosine phosphatases which in turn suppresses defense mechanism of the infected macrophages [53]. In two recent findings, *L. donovani* and *L. amazonensis* was shown to transcriptionally inhibit the components of inflammasome in infected macrophages [52, 54]. Though inflammation activation might not be fully blocked by the pathogens, but the magnitude or extent of inflammasome activation is surely noticeably limited by *Leishmania* as compared to other infectious pathogens like bacteria or protozoan parasites like *Trypanosoma cruzi* and *Toxoplasma gondii* [4].

#### **3.3 The NLRP3 inflammasome and its activation during** *Leishmania* **sp. infection**

Initial stage of macrophage infection by *Leishmania* triggers inflammasome activation in the infected macrophages prompting Caspase-1 activation and production of IL-1b. The role of NLRP3 inflammasome in Caspase-1 activation was established by the absence of the process in NLRP3 or ASC deficient cells [55]. Macrophage inflammasome activation restricted parasite multiplication by Caspase-1 and IL-1b production and stimulation of ROS generation via p47phox and arachidonic acid-NADPH oxidase signaling pathway in *L. infantum* [56]. In conjunction with these findings, it was revealed that *L. amazonensis* triggered Dectin-1 activation, which resulted in Syk kinase activation and ROS production by NADPH oxidase, a step required for NLRP3 activation in macrophages [57]. Furthermore, inhibiting ROS and NADPH oxidase during the primary infection was sufficient to prevent inflammasome activation, demonstrating that the earliest signals for inflammasome activation occur during parasite phagocytosis [57]. These findings highlight the role of ROS in the activation of NLRP3 during *Leishmania* infection. Lipophosphoglycan (LPG) from several *Leishmania* species has recently been demonstrated to activate caspase-11 in macrophages, suggesting non-canonical NLRP3 inflammasome activation [58]. It was also observed that mitochondrial phosphatase phosphoglycerate mutase family member five, a protein involved in restricting *Leishmania* multiplication in macrophages, is required for the production of IL-1b in response to *Leishmania* infection, implying that this protein is involved in the NLRP3 inflammasome activation [59]. Compounds that activate inflammasomes and promote IL-1b production, such as polyester poly (lactide-co-glycolide acid) nanoparticles infused with an 11 kDa *Leishmania* antigen, the antiprotozoal drug diterpene kaurenoic acid, and the conventional anti-leishmanial drug Amphotericin B, have also been shown to limit parasite replication in macrophages [60, 61]. In a visceral Leishmaniasis model, anti-IL-1b treatment inhibited parasite clearance by Amphotericin B. These investigations show indisputably that the inflammasome is activated as a result of *Leishmania* infection and plays a key role in limiting proliferation of *Leishmania* in macrophages.

#### **4.** *Leishmania* **evades host defense**

#### **4.1 Curbing inflammation**

To counter host defenses, *Leishmania* exploits a multitude of intervention mechanisms by targeting various signaling pathways in macrophages. Even before the parasites are inoculated by the sand fly vector, the proteophosphoglycan rich secretory gel produced by the parasites promotes *L. mexicana* viability by increasing alternative activation and arginase activity in the macrophages [18]. Interestingly, *Leishmania* amplified the production of the proinflammatory cytokines IL-1, TNF, MIP-1, and MCP-1 as well as the anti-inflammatory cytokine IL-10 induced by leishmanial LPS [62]. Murine peritoneal macrophages infected with *L. donovani* promastigotes has recently been observed to stimulate expression of PPARγ of the host, which is believed to suppress inflammation and guard the host from irreparable damage. The parasite was easier to remove when PPAR was inhibited [63]. *Leishmania* also activated host PTPs (protein tyrosine phosphatases) such as PTP1B, TC-PTP, PTP-PEST, and SHP-1. PTP activation causes a wide range of beneficial events for the parasite, including a decrease in proinflammatory processes, a decrease in IL-12, NO, TNF, phagolysosomal maturation, and antigen presentation by class II MHC molecules [64, 65]. TRAF3 is another newly discovered target of *L. donovani* promastigotes. In RAW 264.7 cells and bone marrow-derived macrophages, the parasite blocked TRAF3 (TNF receptor-associated factor) degradation to impede TLR4-mediated inflammatory response in the host. TLR4 activation requires TRAF3 degradative ubiquitination. TRAF3 knockdown by shRNA reduced parasite load [66]. The preceding investigations demonstrate the wide range of host targets that *Leishmania* uses to avoid macrophage activation and the consequent proinflammatory response. As *Leishmania* develops infection and multiplies within the macrophage, the macrophage may ultimately undergo apoptosis. The parasite postpones apoptosis of macrophage but eventually uses the apoptotic cell to propagate to nearby non - infected macrophages with limited access to extracellular immune recognition systems. Hence, when it is at its most susceptible phases, *Leishmania* skillfully manipulates its host to escape immune detection and associated inflammation.

#### **4.2 Interfering with host cell signaling**

*Leishmania*-infected macrophages lack events associated with activation and are insensitive to IFN-γ [51]. It has also been found that IFN-γ receptors are downregulated by *L. donovani* promastigotes and promote the expression of the cytokine signaling suppressor SOCS3 [67]. As a result, *L. donovani* promastigotes may effectively turn off the main signaling cascade of one of the most essential macrophage activators*. L. donovani* amastigotes, like promastigotes, suppressed IFN-induced MHC class II and iNOS expression. Infection with *L. donovani* amastigotes, on the other hand, reduced IFN-induced gene expression without changing STAT1 activation. Rather, amastigotes decreased IFN-induced nuclear translocation of STAT1 through interfering with STAT1's interaction with karyopherin importin-α5 [68]. The fundamental mechanics are yet unknown.

#### **4.3 Avoiding oxidative damage**

The reactions of *Leishmania* to oxidative stress is different for different *Leishmania* species and host cell they infect [69]. For instance, peritoneal macrophages infected with *L. major*, when elicited by thioglycolate, triggered ROS generation by stationary

*The Tale of Mastering Macrophage Environment through the Control… DOI: http://dx.doi.org/10.5772/intechopen.102671*

phase promastigotes whereas the same was inhibited in macrophages infected with *L. amazonensis* [70]. To combat oxidative stress, various Leishmania species employ a variety of strategies. *L. donovani* axenic amastigotes, for example, were able to reduce both intracellular and mitochondrial ROS by inducing mitochondrial uncoupling protein 2 (UCP2) upregulation, hence ROS inhibition [53]. *Leishmania* can also escape oxidative damage by inhibiting the assembly of phagolysosomal membrane NADPH oxidase and the formation of ROS within the parasitophorous vacuoles [71]. The parasite's insertion of the surface glycolipid lipophosphoglycan (LPG) in the phagosomal membrane may further hinder recruitment of the NADPH oxidase components to the parasitophorous vacuoles [30]. In addition to directly damaging the parasite's oxidative damage caused by ROS promotes macrophage apoptosis, destroying the parasite's replicative niche.

#### **4.4 Countering antigen presentation**

Antigen cross-presentation is a significant aspect in pathogen immunity. It entails presenting phagocytosed cargo-derived foreign proteins on class I MHC for cytotoxic CD8+ T cells recognition and a systemic immune response coordination. The macrophage, as a specialized antigen presenting cell (APC), contributes in the cross-presentation of proteins generated from *Leishmania*. In macrophages infected with *L. donovani* or *L. major*, the parasite strategically escapes immune responses offered by the host by inducing SNARE VAMPS cleavage, thus preventing antigen cross-presentation [34]. No such inhibition is observed in *L. donovani* amastigote infected macrophages. VAMP8 disruption inhibited assembly of NADPH oxidase, resulting in more effective phagosomal acidification and proteolysis, and thereby reducing presentation by MJHC class I and activation of T cells [71–73]. The parasite also inhibited antigen cross-presentation by disrupting membrane lipid microdomains [74]. Indeed, infected cells had lower membrane cholesterol levels, and the defect in antigen presentation could be repaired with liposomal administration of exogenous cholesterol. Liposomal cholesterol was also found to be enhancing ROS and RNI degeneration, as well as expression of proinflammatory cytokine and intracellular parasite death, and was linked to cellular stress and ROS-induced apoptosis in peritoneal cells infected with *L. donovani* promastigotes [75].

#### **4.5 Inducing autophagy**

*L. amazonensis* amastigotes and stationary phase promastigotes increased intracellular parasite survival by autophagy induction in peritoneal exudate cells or macrophages derived from bone marrow. Autophagy inhibitors like 3-methyladenine (3MA) or wortmannin lowered parasite burden, but autophagy inducers like rapamycin or starvation had little effect or increased parasite load [76, 77]. Autophagy induction was related with a decrease in NO, emphasizing the importance of this mechanism in infection establishment.

#### **4.6 Exploiting macrophage environment to activate its antioxidant defense mechanism through cyclic nucleotide signaling pathway**

Following phagocytosis by macrophages in the early stages of infection, the parasites are subjected to severe oxidative stress as a result of a respiratory burst offered by the macrophages releasing ROS and RNS [78, 79]. Superoxide dismutase, peroxidoxin, and trypanothione reductase are three *Leishmania* components involved in antioxidant defense against ROS and RNIs. The parasites are rendered

susceptible to death in the ROS producing macrophages upon the disruption of the above-mentioned genes [80–83]. Furthermore, resistance against oxidative damage is induced when the organism is pre-exposed to environmental stress [78, 84]. In many unicellular eukaryotes, the cAMP response has been linked as one of the key environmental sensing machineries related with environmental stress response. Despite the fact that *Leishmania* parasites are subjected to extreme stress, which is a trigger for differentiation, and the relevance of cAMP in parasite differentiation has been documented, knowledge about the function of cAMP in *Leishmania* pathogenicity and resistance against oxidative stress is still obscure. A high temperature of 37°C, a low pH of 5.5 and nutritional starvation, all these collectively trigger cell cycle arrest in promastigote resulting in differentiation into aflagellar amastigote forms inside the macrophage. Occurrence of the major antioxidant gene upregulation was confirmed by analyzing the mRNA expression of these genes under stressed condition and maximum elevation of Ldpxn1 level was found indicating the activity of antioxidant genes in *Leishmania* during stress [8]. Not only that, antioxidant gene protein upregulation was also observed in *Leishmania* treated with cAMP analogues, suggesting a crucial role of cAMP in promoting antioxidant defense in *Leishmania*. Chemical or genetic inhibition of intracellular cAMP resulted in a decrease in the mRNA and protein levels of these anti-oxidant genes. The role of cAMP in parasite infectivity was reinforced by the discovery that promastigotes with a steadily overexpressed pde gene had a lower ability to infect IFN-γ activated macrophages than normal cells. The cAMP response is most likely one of several environmental sensing tools connected with *Leishmania* differentiation, and many other biochemical reactions may collaborate with cAMP in the process of differentiation resulting in parasite transformation.

#### **5. Role of cAMP in survival and infectivity of the parasites**

In eukaryotes, cAMP, a second messenger which is formed from ATP by the membrane- bound enzyme, receptor adenylate cyclases (RAC), is a key component that controls a wide range of cellular activities such as cytoskeletal modeling, cell proliferation, virulence, cellular differentiation, and death [85]. There have been reports of various isoforms of both membrane-bound receptor adenylate cyclases and soluble adenylate cyclases in *Leishmania* [86]. cAMP has been found to be involved in signal transduction processes that occur during transformations not only in *Leishmania*, but also in other similar kinetoplastid protozoa. In *Trypanosoma brucei* [87] and *Trypanosoma cruzi* [88], various life cycle phases have variable intracellular cAMP concentrations. Owing to the importance of cAMP and the role it plays in the transformation of kinetoplastid parasites, receptor adenylate cyclases in *L. donovani* have further been investigated. In *L. donovani*, the occurrence of receptor adenylate cyclase (RAC) has also been revealed, and a membrane associated RAC-A has been established to be functional when exposed to phagolysosome conditions (PC) actively catalyzing cAMP production [89]. In addition to the direct action of LdRAC-A in stress-induced intra-cellular cAMP generation, intracellular PPi and pyrophosphatases also perform a significant role in modulating cAMP concentration in the cell. A higher PPi concentration might be modulating adenylate cyclase reactions and thus, inhibits cAMP generation in the parasites (**Figure 1**) [90]. Aside from pyrophosphatases, which control intracellular cAMP synthesis by receptor adenylate cyclases, other aspects of cAMP regulation must be investigated. Intramacrophage cAMP concentration is increased upon infection-induced Prostaglandin E2 production in

#### *The Tale of Mastering Macrophage Environment through the Control… DOI: http://dx.doi.org/10.5772/intechopen.102671*

the *Leishmania*-infected macrophage [91]. Intracellular cAMP level is maintained by phosphodiesterases (PDEs) which are universal enzymes that hydrolyze cAMP to 5′-AMP or cGMP to 5′-GMP to terminate the cyclic nucleotide signaling pathway, are the only mechanism for the cell to get rid of the cAMP generated for managing various cellular activities [92]. Cytosolic PDE activities decrease throughout stage differentiation, although the activity of membrane bound PDEs remains constant. This data suggests that PDEs have a crucial function as a regulating factor during stage differentiation of *Leishmania* [93]. Not only membrane-bound phosphodiesterases, but cytosolic phosphodiesterase, PDED, is also important in the regulation of cAMP homeostasis by enhanced hydrolytic property through PKA-mediated activation of PDED [94]. PDED plays a role in the regulation of PKA activity and in turn maintains cAMP homeostasis in the parasite when initially exposed to stress conditions.

Despite the fact that cAMP-dependent protein kinase (PKA) exists and functions in eukaryotes, the role of PKA in cAMP signaling in this specific parasite is still ambiguous. PKA, being the first downstream effector of cAMP in the RAC pathway, facilitate γ-P transfer to particular ser/thr residue from ATP [95]. Upon exposure of *Leishmania* to environmental stress, a significant upregulation of PKA activity, concomitantly with cAMP level increase, is observed, suggesting a connection between cAMP and PKA during parasite differentiation. PKA activity of five distinct *Leishmania* species was shown to be relatively high in logarithmic, as well as stationary phase promastigotes, with *L. amazonensis* being the most active and *L. donovani* being the least active [96]. A regulatory subunit of cAMP-dependent PKA, LdPKAR1, have been identified in *L. donovani* which plays significant role in metacyclogenesis, intra-macrophage survival and enhanced infectivity of the parasite [97]. Thus, cAMP is emerging as a key regulator of cytological processes in kinetoplastida parasites [98]. Despite the discovery of multiple cyclic nucleotide binding effector molecules, there is still lack of complete understanding on cAMP signaling from receptor to effector.

#### **6. Conclusion**

Being an intracellular parasite, *Leishmania* has adopted various ways to enter the host cells efficiently and then subsequently try to neutralize the hostile environment of macrophages. Although various strategies have been adopted by this parasite to establish successful infection, we are mainly concerned about stressing the role of inflammasome activation in host cells and cAMP homeostasis of the parasite in this chapter. To summarize, it can be stated that recent advances in research have increased our understanding of the role of cAMP signaling in kinetoplastid parasites such as *Leishmania* and its relationship to parasite infectivity. These findings shed light on the activity of many enzymes involved in cAMP metabolism. It can be suggested that modulating cAMP levels in the parasite might be one of the methods for controlling leishmaniasis, and that the molecules involved could be explored as powerful therapeutic targets against the leishmaniases.

#### **Acknowledgements**

We would like to thank Personal Research Grant (PRG), University of Kalyani, DST-PURSE, University of Kalyani and INSA Senior Scientist Fellowship for contingent costs associated with the drafting of this chapter.

## **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Anindita Bhattacharjee1 , Arunima Biswas1 \* and Pijush K. Das2 \*

1 Molecular Cell Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani, India

2 Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, Kolkata, India

\*Address all correspondence to<EMAIL_ADDRESS>and<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Tale of Mastering Macrophage Environment through the Control… DOI: http://dx.doi.org/10.5772/intechopen.102671*

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#### **Chapter 7**


**6. Plant metabolites**

*Livestock Health and Farming*

this basis are:

used are listed as follows:

ruminants [45].

which do not compete with human feeding. Therefore, diverse advantages can be observed when utilizing alternative forages such as (a) a considerable reduction in the feeding source costs; (b) exploitation of nutrients contained in agriculture by-products which otherwise would not be used (these by-products could be high in rapidly digestible carbohydrates or in fiber, both suitable for ruminants); and (c) an increase in the by-product cost which eventually will create economic benefits for producers and the productivity chain supply. Moreover, some agricultural by-products do not need any processing to be offered as animal feeding, hence the desirability of these by-products. Some of the ruminants feeding produced under

a.Crop by-products such as garlic leaves, onion leaves, cocoa husks, coconut meal, cracked rice, sugarcane bagasse, molasses, tapioca discards, oat straws,

b.Perennial crops, seeds, and leaves of shrubs and trees like *Leucaena*, gua-

Some farmers are still not aware of the nutritional value contained in byproducts or in the form to be included into the productivity chain of animal feeding in an efficient way. In this regard, Asia and Africa are heavily focused on attempting to reach this goal. Due to the nature of some agricultural by-products, these tend to decompose in a short time.  Therefore, some of the preservation techniques commonly

I. *Silages*: Many of the agricultural by-products are obtained in huge quantities due to the nature of the crops. However, the high humidity contents contribute to a short lifetime due to the rapidly appearance of fungi and, eventually, a decomposition. Therefore, the silage elaboration is a recommended preservation method due its large periods of storage, and it can always be offered fresh and with certain aroma provided from the fatty acids synthetized in the lactic fermentation which will add palatability for

II.*Chemical treatments*: Some agricultural residues obtained from cereal crops are treated with chemicals to increase their digestibility. Thus, by-products with high lignocellulosic complexes could be treated with ammonia in anaerobic conditions to enhance lignin and fiber hydrolysis, which will improve their digestibility [46]. However, this process requires special plastic sheets that increases costs and could become an unaffordable process for small producers. In this way, previous researches have reported diverse alternatives using mud and eliminating the use of plastic sheets [47].

III.*Multi-nutritional blocks*: Another conservation technique which involves the utilization of high humidity agricultural by-products is the elaboration of multi-nutritional blocks [48]. This technology is very flexible and allows the producer to use ingredients considered as indispensable in animal feeding. Additionally, important nutrients could be available for longer periods of time since the useful life of these blocks is very extensive. Although in dry seasons, drought decreases considerably the nutritional quality of forages. These blocks are generally offered as supplementation in livestock feeding in rangelands as part of an extensive feeding system, and they are commonly

and some aquatic crops like water hyacinth and azolla [38–41]

muchil, mesquite, mango, ebony, etc. [42–44]

**124**

In the last years, ruminants have been target of several feeding strategies aiming to reduce ruminal methane production and emissions; most of them have been stated earlier in this chapter. However, the use of secondary metabolites arises as a viable and newer alternative in this concern. There is evidence which proves certain secondary metabolites, such as condensed tannins, saponins, and alkaloids, reduce methane production in in vivo and in vitro assays [54]. Generally, the mechanisms of action of these compounds point out to certain metabolic pathways:

I.**Tannins**: Tannins are water-soluble polyphenol polymers with a high and diverse molecular weight. They can form complexes with proteins, mainly, and metal ions, amino acids, and polysaccharides in a lesser extent. These metabolites are normally synthetized in shrubs, trees, legumes, fruits, cereals, and grains [55]. Tannins are divided into two groups: condensed tannins (CTs) and hydrolysable tannins (HTs).

*Hydrolysable tannins*: These are complex molecules attached to a polyol group as a central core which are partially or fully esterified with a phenolic group (e.g., gallic acid). The remaining phenolic groups could be later esterified or oxidized to produce more complexes with HTs [55].

*Condensed tannins*: These compounds are also known as proanthocyanidins and are mainly polymers of the flavan-3-ol units which are bind by interflavonoids C3-C8 and C4-C6 linkages, such as catechin and epicatechin. The methanogenic activity conferred to tannins is mostly due to the condensed tannins; CTs attach to proteins and avoid their degradation in rumen. Additionally, CT decrease methanogenesis through a reduction in fiber digestion [56]. Some studies affirm that CT enhances acetate formation via acetogenesis; this metabolic pathway uses hydrogen for acetate synthesis and reduces methanogenesis [57].

II. **Saponins**. According to their chemical structure, they are divided into two groups: steroids and triterpenoids. Steroids are predominantly in

plants and are composed of 27 carbon atoms in the central skeleton of its molecule (e.g., spirostanol and furostanol). Otherwise, triterpenoids are composed mainly of aglycones with 30 carbon atoms in its molecule (e.g., oleanane) [58]. These are the most common types of saponins, especially in legumes [59]. Methanogenic action of saponins occurs by protozoa defaunation which is associated to methanogens. Moreover, saponins enhance production of propionate, a natural competitor of methane in hydrogen capture [58]. Nevertheless, some studies affirm that methane inhibition action by saponins is dose and time dependent and not conclusive [59].

III.**Flavonoids**. Flavonoids are phenolic compounds (like tannins); however, these contain only 15 carbon atoms linked to 2 aromatic rings connected through a 3-carbon bridge [60]. These metabolites are particularly studied for human purposes, and their biological benefits to health correlated to their consumption [61]. Almost all flavonoids are conjugated to glycosides and are common to find hydroxyl groups in carbons with four, five, and seven positions [60]. In addition, flavonoids stimulate microbial metabolism and reduce methane production through enhancing acetogenesis pathway and increasing hydrogen capture in propionate anabolism, in a similar way as described earlier with saponins [28, 59].