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									         Cytokines, key players to turn on/off the anti-Trypanosoma cruzi innate
         defense mechanisms
         Eugenio A. Carrera-Silva1, Susana Gea2 and Natalia Guiñazú3*
        1 Department of immunobiology, School of Medicine, Yale University. New Haven, USA.
        2 CIBICI-CONICET, Universidad Nacional de Córdoba. Haya de la Torre y Medina Allende s/n, 5000. Córdoba,
        Argentina.
         3* IDEPA-CONICET, Universidad Nacional del Comahue. Buenos Aires 1400, 8300. Neuquén, Argentina.

         The early host resistance against Trypanosoma cruzi infection depends on a complex interplay among cytokines, chemical
         mediators and cells. The major innate immune mechanism against intracellular parasites in phagocytes relies on the
         production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The initial step for ROS production is
         the generation of superoxide anion catalyzed by the enzyme NADPH oxidase. The phagocyte´s NADPH oxidase is a
         multiprotein complex, which exists in the dissociated state in resting cells and assembles into the functional complex upon
         stimulation. Additionally, the high amount production of the RNS nitric oxide (NO) depends on the enzyme nitric oxide
         synthase induction by cytokines. The combination of superoxide anion and NO yields peroxynitrite, the most parasite-
         harmful reactive species. The anti-inflammatory and pro-inflammatory cytokine balance modulates the activation and
         induction of both enzymes. Here we discuss the cellular processes involved in macrophage-mediated host defense against
         Trypanosoma cruzi, and the implications of ROS, RNS and cytokine regulation in host resistance.

         Keywords Cytokines, NADPH oxidase, Nitric oxide synthase, Phagocyte, Trypanosoma cruzi.



         1. Activation of innate immune response and microbicidal mechanisms
The adequate activation of innate immune response is essential to control the invading pathogens and to maintain
homeostasis. In the past decade, there have been important advances in our understanding of how the inflammatory
response to pathogens is initiated. Thus, a typical inflammatory response consists of four components: inflammatory
inducers, which initiate the inflammatory response and are detected by sensors. Sensors of innate immunity, such as
Toll-like receptors (TLRs), are expressed on specialized sentinel cells including tissue-resident macrophages, dendritic
cells, and mast cells. When innate immune receptors are triggered, they induce the production of mediators, including
cytokines, chemokines, bioactive amines, eicosanoids, and products of proteolytic cascades, such as bradykinin. These
inflammatory mediators act on various target tissues to elicit changes in their functional states that optimize adaptation
to the noxious condition. The acute inflammatory response is normally terminated once the triggering insult is
eliminated, the infection is cleared, and damaged tissue is repaired [1].
   The type of response induced under a given condition depends on the nature of the inflammatory trigger. For
example, bacterial pathogens are detected by tissue-resident macrophages and induce the production of inflammatory
cytokines (e.g., TNF, IL-1, IL-6) and chemokines (e.g., CCL2 and CXCL8), as well as prostaglandins. Viral infections
induce the production of type-I interferons (IFNα, IFNβ) by infected cells and the activation of cytotoxic lymphocytes,
whereas infections with parasitic worms lead to the production of histamine, IL-4, IL-5, and IL-13 by mast cells and
basophils. Cytokines are fundamental to induce microbicidal mechanisms against pathogens and to drive the adaptive
immune response as well [1- 3].
   Macrophage role is pleiotropic and include antigen presentation, target cell killing, regulation of inflammatory
response, removal of cell debris and promotion tissue remodeling. These functional responses are dependent on micro-
environmental signals. A key immunological function of phagocytes is to control invading microorganisms. This
property lies on the capacity to generate high amounts of reactive oxygen species (ROS) and reactive nitrogen species
(RNS). ROS are oxygen-derived small molecules, such as superoxide anion (O2-), hydroxyl radical and anion (OH and
OH-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), while RNS are nitrogen-containing oxidants such as
nitric oxide (NO) and peroxynitrite anion (ONOO-). ROS and RNS are unstable and rapidly interact with a large
number of target molecules including proteins, lipids, carbohydrates, and nucleic acids. ROS and RNS through such
interactions may irreversibly destroy or alter the function of the targeted molecule [4].
   This chapter will discuss the activation mechanisms of some enzymes responsible of ROS and RNS generation, the
contribution of anti- and pro-inflammatory cytokines. The main microbicidal mechanisms involved in the control of
Trypanosoma cruzi acute infection will be reviewed.

         1.1    NADPH oxidase
Phagocytes such as polymorphonuclear neutrophils (PMN), eosinophils, monocytes and macrophages constitute one of
the most powerful means of host defense against microbes [5]. Upon infection and inflammation, leukocytes migrate
towards the infection site attracted by chemoattractants such as the complement fraction C5a, the N-formyl-methionyl-
leucylphenylalanine (fMLF) peptide, IL-8, platelet activating factor (PAF) or leukotriene B4 (LTB4). At the infection
site, phagocytes recognize and engulf the microbe. After internalization, phagosomes subsequently fuse with
intracellular granules to form the phagolysosome, within which microbial killing is achieved by a combination of non-
oxidative and oxidative mechanisms. Potent non-oxidative killing mechanisms include antimicrobial peptides (cationic
proteins, defensins, lysozyme, etc). The oxygen-dependent killing mechanism involves the generation of ROS. Together
these different mediators contribute to the elimination of the internalized microorganism [6].
   ROS are produced by phagocytes in a potent "oxidative burst", characterized by a rapid increase in oxygen uptake, an
increase in glucose consumption and abrupt ROS release. The enzyme responsible for superoxide anion (O 2-)
production is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. NADPH oxidase enzyme uses
electrons derived from intracellular NADPH to generate O 2-. Much of the O2- produced is dismuted by superoxide
dismutase to produce oxygen and hydrogen peroxide (H2O2) (Figure 1), which within neutrophils, is converted by
myeloperoxidase to hypochlorous acid and chloramines inside the phagosome. All the products of the oxidative burst
are part of the strong microbicidal system of phagocytes [7].

                                                   e        H2O2
                                                xid e
                                             ero as                            Fig. 1 Schematic representation of ROS formation from
                                           up mut
                                          S is            Hydrogen
                                           D              peroxide             superoxide anion. NADPH oxidase complex reduces oxygen
                                                                               (O2) and generate superoxide anion (O2-) using NADPH as an
            NADPH                                                              electron donor. Much of the O2-- is subsequently transformed
            oxidase                            Iron
     O2
                                O2-
                                                         OH- + OH
                                                                               into other ROS. By superoxide dismutase to produce
                                                                               hydrogen peroxide (H2O2), in the presence of iron H2O2 is
   Oxygen
                          Superoxide                    Hydroxyl anion         transformed to hydroxyl anion and radical (OH- and OH) via
                            anion Ni                      and radical
                                     tric                                      the Fenton reaction and the Haber-Weiss reaction.
                                               oxi
                                                  de
                                                           OONO-
                                                                               Superoxide anion in the presence of nitric oxide (NO) reacts
                                                                               to yield peroxynitrite anion (OONO-).
                                      Nitric oxide       Peroxynitrite
                                       synthase

   On the other hand, it has been reported that O2- is also produced in a NADPH-dependent manner in non-phagocytes
cells. Because gp91phox (NOX2) is not expressed in these cells, the presence of non-phagocyte-type NOX2 homologs
were found in virtually all tissues and have been implicated in many areas of biology, including cell growth, apoptosis,
and cancer, innate immunity and inflammation, angiogenesis and blood pressure regulation, cell signaling and motility,
and hormone synthesis. To date, five NOX isoforms (NOX1, NOX2, NOX3, NOX4, and NOX5) and two related
enzymes, DUOX1 and DUOX2, were reported. The NOX family members are now recognized as producers of low
levels of ROS that play critical roles in maintaining normal physiologic processes and in the etiology of multiple
diseases [8].
   NADPH oxidase is a multicomponent enzyme that in leukocytes is comprised by the cell-membrane-bound
gp91phox (NOX2) and p22phox proteins (which together constitute the flavocytochrome b558), and the cytoplasmic
proteins p47phox, p67phox, p40phox, and Rac. NADPH oxidase is dormant in resting cells but becomes active when
cells are stimulated (Figure 2).
                                                                                                     2O2                  2O2-
              Extracellular space                                              Extracellular space                                H+
              or phagosome                                                     or phagosome



                                                gp91     p22                                                gp91         p22

              Intracellular                                                    Intracellular          Rac GTP
                                                                                                                          p47
              space                                                            space                        p67
                                                                     Rac GDP                                       p40
                                                                 Rho GDI
                                              p47                                               NADPH                          NADP+
                              p67
                                       p40


Fig. 2 Schematic representation of resting and activated phagocyte NADPH oxidase. The proteins gp91phox and p22phox are
associated at the plasma membrane and stabilize each other. NADPH oxidase activation is regulated by the organizer p47phox and
activator p67phox subunits, and requires GTP-bound Rac. The cytosolic subunits p47phox, p67phox and p40phox are preassembled
in the cytosol and upon activation translocate to the membrane, where gp91phox and p22phox are located. In resting cells, Rac is
found in a GDP-bound state stabilized by RhoGDI. With stimulation, Rac translocates to the membrane independently of p47phox
and p67phox. GTP-bound Rac interacts with p67phox. After the phagocyte cell is activated through the action of soluble
chemoattractants and chemokines, or phagozitable particles, the stimulated NADPH oxidase enzyme catalyzes the one-electron
reduction of oxygen to produce superoxide anion by using NADPH as substrate. This enzyme uses electrons derived from
intracellular NADPH to generate superoxide anion, which subsequently is transformed into other ROS.
   In the cytoplasm of resting cells are two protein complexes, one composed of p47phox, p67phox and p40phox and
the other containing Rac and RhoGDI, the GDP dissociation inhibitor for Rho. Almost 100% of p47phox is located in
the cytosol alone or in a complex containing equimolar of p67phox and p40phox [9, 10]. Upon activation, p47phox is
heavily phosphorylated, enabling it to co-localize with the flavocytochrome b558 at the membrane [11]. The p47phox
protein is believed to be responsible for the transport of the cytosolic subunits to the membrane and is thus considered
to be the organizing adaptor protein [12, 13]. On the other hand, p67phox represents an essential activating cofactor,
possessing a domain that regulates the reduction of FAD by NADPH [14]. During activation, p67phox and p40phox
also become phosphorylated [8]. The significance of p40phox phosphorylation is unknown, although it has been
reported to promote negative regulation of NADPH oxidase function by p40phox [15]. The translocation of Rac to the
plasma membrane NADPH oxidase in human neutrophils occurs through an independent mechanism from, but in a
coordinated manner with, translocation of the p47phox/p67phox complex [16].
   NADPH oxidase physiological activator stimuli include phagozitable particles such as bacteria and yeast, certain
molecules that induce chemotaxis in these cells, certain bioactive lipids, complement proteins and antibodies. The
stimuli of highest importance are phagozytable particles. Coating the surface of a pathogen, by antibodies or
complement proteins, to enhance phagocytosis is called opsonization. Antibodies do this in either two ways. In the first,
bound antibodies coating the pathogen are recognized by Fc receptors on phagocytic cells that bind to the antibody C-
region. Alternatively, antibodies binding to the surface of a pathogen can activate the complement system. Complement
proteins binding to the pathogen surface; opsonize the pathogen by binding complement receptors on phagocytes.
Phagocytosis is an active process, in which the bound pathogen is internalized in a membrane-bounded vesicle, known
as phagosome or endocytic vacuole. The lysosome liberates its content into the phagosome and generates the
pahgolysosome. NADPH oxidase enzyme assembles at the phagosomal membrane. Once activated, O 2- production
occurs inside of the phagosome. The isolation of superoxide release into the phagosome is important since ROS are able
to damage host tissues too.
   Other non-physiological molecules also activate NADPH oxidase complex and have been important in understanding
the regulation of the phagocyte NADPH oxidase. In this sense, PMA and calcium ionophores induce superoxide
production, which does not have to be initiated through membrane receptors (PMA activates PKC, calcium ionophores
increase the intracellular calcium concentration).

         1.2   Nitric oxide synthase

Nitric oxide (NO) is a messenger molecule functioning in several processes, such as neurotransmission, vascular
regulation and host immune defense, among others. NO is produced by the oxidation of L-arginine by the enzyme NO
synthase (NOS). Three NOS isoforms have been reported, neuronal NOS (nNOS), endothelial NOS (eNOS), and
inducible NOS (iNOS). Both nNOS and eNOS are constitutive and Ca2+-dependent, while iNOS isoform is calcium-
independent and inducible. NOS isoforms not only produce NO, but also a number of species resulting from oxidation,
reduction, or adduction of NO in physiological milieus, thereby generating various nitrogen oxides, S-nitrosothiols,
peroxynitrite (ONOO-), and transition metal adducts [17]. An important protective role for RNS has been established in
macrophage killing of intracellular protozoa [18- 21], bacteria [22], fungus [23] and viruses [24]. A similar role has
been demonstrated in neutrophil killing of Candida albicans [25, 26] and Staphilococcus aureus [27].
The aminoacid L-arginine is the common substrate of two enzymes NOS and arginase. Arginase enzyme metabolizes L-
arginine to produce urea and L-ornithine. In subsequent steps, L-ornithine is metabolized by ornithine decarboxilase
(ODC) to produce polyamines (putrescine, spermidine, spermine) and proline, which promote cell growth and collagen
production. Arginase exists in 2 isoforms, arginase I enzyme is located in the cytosol and expressed in the liver as one
of the enzymes of the urea cycle which detoxifies ammonia in mammals, and arginase II protein is expressed as a
mitochondrial protein in a variety of peripheral mammalian tissues, most prominently in kidney, prostate, small
intestine and the lactating mammary gland [28]. The reciprocal regulation of iNOS and arginase has been reported in
various inflammatory pathologies. Arginase activity or expression induction was demonstrated in murine inflammatory
cell infiltrates in schistosomiasis [29], american trypanosomiasis [30, 32], leishmaniasis [33, 34], viral [35, 36] and
bacterial [37, 38] infections.
   Since the availability of intracellular L-arginine is a rate-limiting factor in NO production, arginase activity may
down-regulate NO production by competing with NOS for L-arginine. While in macrophages stimulated by
inflammatory Th1 cytokines TNF-α and IFNγ, iNOS is induced, arginase expression in murine macrophages is induced
by Th2 cytokines IL-4, IL-10 and IL-13 [39]. In murine disease models that follow the Th1/Th2 paradigm with regard
to disease susceptibility or resistance iNOS is induced in the context of a Th1-dominated resistant phenotype while
macrophage arginase is up-regulated during Th2-mediated disease progression. In contrast to its involvement in host-
detrimental immunopathology, myeloid cell arginase can also serve a crucial host-protective function by down-
regulating excessive Th1-induced inflammation [28].
         2. Regulation of microbicidal mechanisms by cytokines
In the course of infectious diseases, a permanent challenge is imposed on the immune system: to eliminate, or at least to
control, the infectious agent and to minimize the destruction of tissue architecture. The extent of the immune response
and the action of potentially harmful effector cells should be tightly controlled, either by regulatory cytokines or by the
elimination of lymphocytes by apoptosis. Anti-inflammatory response, mediated by IL-10 and TGF, counteract the
effects of inflammatory cytokines such as IL-12, TNF and IFN [40]. In this sense, macrophages are innate immune
cells with well-established roles in the primary response to pathogens, but also in tissue homeostasis, coordination of
the adaptive immune response, inflammation, resolution, and repair. They can function as control switches of the
immune system, securing the balance between pro- and anti-inflammatory reactions. For this purpose and depending on
the activating stimuli, these cells can develop into different subsets: classically (M1) or alternatively (M2) activated [41-
43].
   The classical activation profile occurs in a type I cytokine environment (IFNγ, TNF) or upon recognition of
pathogen-associated molecular pattern (PAMPs) such as LPS, lipoproteins, dsRNA, lipoteichoic acid, etc, and
endogenous danger signals such as heat shock proteins, etc. As such, they play an important role in protection against
intracellular pathogens, and under certain conditions also cancer cells. Classically activated macrophages (M1) typically
produce high levels of IL-12 – and in humans also IL-23 [44] – and low levels of IL-10 and are consequently strong
promoters of Th1 immune responses. In addition, these cells exert anti-proliferative and cytotoxic activities, resulting
partly from their ability to secrete RNS and ROS and inflammatory cytokines (TNF, IL-1, IL-6) [45- 46].
Alternative pathway of macrophage activation may arise in a Th2 cell-biased environment with a stereotypic signature
of expressed genes induced by IL-4 and IL-13. As opposed to M1, M2 promote Th2 responses, resulting in effector
functions such as parasite killing and encapsulation, but also exacerbation of Th2 cytokine-associated pathologies, such
as allergies. These macrophages secrete copious amounts of anti-inflammatory molecules like IL-10 and TGF, thereby
down-regulating inflammatory processes initiated by M1. Additionally, M2 exerts selective immunosuppressive
functions in T lymphocyte cell proliferation [47, 48]. Their presence in healthy individuals in the placenta, lungs and
immune privileged sites, as well as in chronic inflammatory diseases like rheumatoid arthritis, atherosclerosis and
psoriasis, further suggests that M2 protect organs and surrounding tissues against detrimental immune responses.
Moreover, they increase the fibrinogenic activity of human fibroblasts [49], promote angiogenesis [50] and wound
repair during the healing phase of acute inflammatory reactions and in chronic inflammatory diseases [41].
   The Arginase 1 enzyme is a signature marker intimately associated to M2 and there has been extensive investigation
of its possible role in alternative versus classical activation [51- 53]. The cross-regulation of the iNOS-arginase balance
by Th1 mediators, such as IFN and LPS, and by Th2 cytokines, such as IL-4 and IL-13, suggests that the measure of
NO level and arginase activity in distinct macrophage populations reflects their activation state, classical or alternative.
However, the distinction between M1 and M2 solely based on the iNOS-arginase balance could be oversimplified.
Other well reported differences between M1 and M2 are situated at the level of cytokine and chemokine secretion, both
in human and mouse [54, 55].
   NADPH oxidases (NOXs) are activated in response to stimulation of associated receptors, such as those for insulin,
platelet-derived growth factor, nerve growth factor, fibroblast growth factor, TNF, the growth factor GM-CSF, and
angiotensin. In phagocytes they are activated by engagement of receptors for the antibody Fc chain (Fcγ) or for
complement during phagocytosis of opsonized particles. NOX2 in neutrophils is also activated by engagement of
receptors for integrins simultaneously with engagement of receptors for soluble agonists such as TNF, formyl
peptides, complement C5a, the growth factors G-CSF and GM-CSF, or macrophage inflammatory protein. NOXs can
be regulated by phosphorylation of p47phox (or TKS5), by flavinloading of gp91phox, by the GTPase activity of Rac1
or Rac2, and by assembly of their components (gp91phox; p22phox; p47phox (or TKS5); p40phox (or NoxO1);
p67phox (or NoxA1); and Rac1 or Rac2) [56].
   The priming of NADPH oxidase is a phenomenon caused by certain molecules that induce a weak oxidative response
or do not directly activate the NADPH oxidase by themselves, but strongly augment superoxide production triggered by
a second stimulus. The priming agents LPS and IFNγ increase the expression level of gp91phox in human monocyte-
derived macrophages and neutrophils [57]. Other cytokines such as G-CSF, GM-CSF and TNFα are able to prime
NADPH oxidase activity in human PMN. The shared mechanism by which GM-CSF and TNFα prime the PMN
respiratory burst involves partial phosphorylation of p47phox [58]. The enhanced phosphorylation of cytosolic
components, decreased inhibition of the autoinhibitory region of p47phox, and assembly of p40phox, p47phox and
p67phox into complexes in the cytosol before translocation to the membrane have been proposed as possible priming
mechanisms [59]. Thus, pro-inflammatory cytokines such as TNF which do not activate NADPH oxidase but prime its
activation in response to a secondary stimulus such as fMLF [60], induce partial phosphorylation of p47phox on Ser345
by ERK1/2 or p38MAPK and promote NADPH oxidase assembly [58, 61].
   Although substantial advances have been made to understand the process leading to NADPH oxidase activation, the
molecular mechanisms involved in NADPH oxidase inactivation are elusive. Deactivation of NADPH oxidase is critical
for timely resolution of inflammation, before bystander tissue damage occurs. Rapid deactivation of NADPH oxidase
could be due to several processes, including receptor desensitization, uncoupling of transduction pathways, and
modulation of the phosphorylation of NADPH oxidase components such as p47phox and p67phox [62]. Important
mediators of this deactivation are also some anti-inflammatory cytokines such as IL-10 and TGF. It has been shown
that IL-10 down-regulates PMN ROS production under certain experimental conditions [62-64]. IL-10 is a pleiotropic
cytokine that plays a critical role in the regulation of inflammatory responses and immune reactions, acting on both
hematopoietic and non-hematopoietic cells [65]. It has been postulated that the inhibition of NADPH oxidase by IL-10,
in microglia cells, is mediated through the inhibition of the JAK1 signaling pathway, which in turn prevents the
translocation of cytosolic subunit p47phox [66], in PMN cells it has been reported that IL-10 inhibits p47phox
phosphorylation through a decrease in ERK1/2 activity [62]. It has been postulated that the deactivating NADPH
oxidase effect of IL-10 could contribute to the tight regulation of the oxidative burst at the inflammatory site. TGF-
β1cytokine one of the most potent endogenous immune modulators of inflammation, has also been implicated in
NADPH deactivation in microglial cells. The molecular mechanism of its anti-inflammatory effect on the activation of
is through the inhibition of NADPH oxidase activity by preventing the ERK-dependent phosphorylation of on p47phox
in microglia to reduce oxidase activities induced by LPS [67].


          3. Trypanosoma cruzi infection control by microbicidal mechanisms
Trypanosoma cruzi is a flagellated parasite that reproduces asexually by binary fission. Like all other trypanosomes, T.
cruzi reside, some stages of their lives, in the blood and/or tissues of vertebrate hosts and during other stages they live in
the digestive tracts of invertebrate vectors. In the mammalian host, in the peripheral blood, the trypomastigotes stage is
found. Inside the cytoplasm of the nucleated T. cruzi replicates as an amastigote [68].
   T. cruzi parasite is the causative agent of Chagas’ disease, and it has been estimated that 10 million people are
infected worldwide, mostly in Latin America where Chagas’ disease is endemic. More than 25 million people are at risk
of the disease. It is estimated that in 2008 Chagas’ disease killed more than 10 000 people [69]. After acute infection,
most patients remain asymptomatic for decades; while a significant fraction of the affected individuals (30%) develop
principally heart and gastrointestinal clinical manifestations during chronic Chagas’ disease. It has been postulated that
T. cruzi persistence together with the immune response induced to multiple myocardial antigens may participate in the
heart damage [70].
   T. cruzi parasite actively invades a variety of cell types such as fibroblasts, muscle cells and macrophages, thereby
triggering a diversity of molecular interactions that mobilize the host immune response. A broad panel of innate
immune cells is the first defense line against T. cruzi, these include neutrophils, tissue macrophages, monocytes,
dendritic cells, eosinophils and natural killer cells [71, 72]. Activated phagocytes play a central role in the control of
infection through the production of ROS and RNS [73, 74]. It has been well recognized that T. cruzi infection augments
the oxidative metabolism of several cell types such as murine spleen cells [75], murine hepatocytes [76], blood
monocytes and macrophages [77, 78], erythrocytes [79], and cardiac cells [80, 81]. It was recently demonstrated that the
derived parasite antigen cruzipain (Cz- is a parasite glycoprotein member of the papain superfamily, expressed as in all
parasite developmental stages) induced ROS production in splenocytes from non-immune and Cz-immune C57BL/6
mice and in a Raw 264.7 macrophage cell line. Cz stimulation favored the production of several ROS, mainly
superoxide anion, and also triggered NADPH oxidase activation [82], this work was the first report unraveling parasite
antigens that might be involved in oxidative stress induced by T. cruzi.
   Besides ROS, it has been reported that T. cruzi infection via inflammatory stimulation induce M1 activated
macrophages that produce NO via the activation of iNOS [20, 21]. Experimental evidence certainly implicates RNS as
important mediators of parasite killing in mice experimentally infected with T. cruzi [73, 74]. NO has been one of the
RNS more frequently studied in the context of T. cruzi infection. In this sense, T. cruzi infection results in elevated NO
levels in plasma [83, 84]. Infected mice which were treated with iNOS inhibitors developed high parasitemia and
mortality [83, 85, 86]. On the other hand, it has been, reported that iNOS deficient mice infected with the Brazil or
Tulahuen strain of T. cruzi are as resistant to infection as wild-type mice. In the absence of iNOS, the infection
resistance would be probably mediated by the enhancement of other immune effector mechanisms. Thus, this work
pointed out to the importance of other microbicidal mechanism than NO alone [87]. In this sense, Alvarez et al (2011)
[74], have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH
oxidase complex to yield superoxide anion. Moreover they demonstrate that the main mechanism responsible of
infection control occurred via the production of NO and superoxide anion simultaneously, which generates
intraphagosomal peroxynitrites.
   Taking into account that T. cruzi has a complex life cycle, several parasite derived-antigens might participate in
microbicidal mechanisms induction. Glycophosphatidylinositol-anchored mucin-like glycoproteins (GPI) [88] and
Tc52-released antigen [89] synergize with IFNγ to stimulate NO production by macrophages. Through mice
immunization models it has been demonstrated that several parasite derived antigens are able to induce the production
of pro-inflammatory cytokines and NO [90- 93]. Currently it remains unclear the host innate receptor that interacts with
some parasite derived antigens proven to trigger an inflammatory response. This is not the case for the Tc52- released
antigen [94] and GPI-anchors of mucin like protein [95] that signal via TLR2. Mice lacking TLR2 display slightly
enhanced susceptibility to T. cruzi in vivo and macrophages show impaired pro-inflammatory cytokine production when
exposed to the parasite in vitro [96]. In addition, there is evidence demonstrating that Cz in vitro up-regulates TLR2
surface expression on immune total spleen and F4/80+ cells. However, it has not been demonstrated whether Cz is able
to interact with TLR2, with further studies required to clarify this issue. Nevertheless, it is probable that Cz modulates
functional responses to the T. cruzi ligands that bind TLR2 [92].
   Moreover, it has been also demonstrated that Cz is able to modulate the iNOS/arginase balance. The immunization of
resistant C57BL/6 mice with Cz primed spleen cells for classical macrophage activation. Cz induced a high level of NO
associated with an up-regulation of iNOS protein and messenger expressions in immune cells. These cells also produced
high levels of IL-12 and IFNγ, probably as a consequence of this pro-inflammatory milieu spleen cell also demonstrated
an amplified NADPH oxidase activation [82, 92]. Altogether these findings indicate that the inflammatory environment
induced by Cz in C57BL/6 background may be participating in the priming of immune cells to a higher production of
RNS and ROS. By contrast, the immunization of susceptible BALB/c mice with Cz resulted in an enhanced anti-
inflammatory (IL-4, IL-5 and IL-10) cytokine secretion, associated with the induction of a CD11b+GR1+ spleen
immature myeloid population that exhibited arginase, but not iNOS activity [97]. Furthermore, Cz-stimulated naive
macrophages secreted IL-10 and TGFβ and displayed enhanced arginase activity which favored T. cruzi growth [98,
99]. In this sense it has been demonstrated that the induction of ODC activity, an enzyme involved in the synthesis of
polyamines, is an essential factors for intracellular parasite replication.
   These reports indicate the importance of parasite derived antigens, the innate receptor triggered, the cytokine milieu
induced as well as the genetic background in the balance parasite resistance/persistence during T. cruzi infection.


         4. The cytokine environment favors Trypanosoma cruzi persistence and immune
         evasion
The coordinated interplay between macrophages, neutrophils and monocytes is crucial for the effective elimination of
noxious agents and the restoration of tissue homeostasis after injury or infection. Initial phagocyte interactions promote
mutual recruitment and antimicrobial activities, but later crosstalk between phagocyte populations dampens pro-
inflammatory responses and promotes resolution of the inflammatory response.
   Although the release of TGFβ, IL-10 and PGE2 can contribute to restoration of homeostasis after tissue injury, these
anti-inflammatory mediators also dampen antimicrobial mechanisms. In this context, it has been shown that
macrophage-mediated clearance of apoptotic cells promotes intracellular parasite persistence, taken advantage of an
immunosuppressive effect. Interestingly, it was reported during parasitic infections by T. cruzi [100], T. gondii [101],
and L. major [102], both short range and systemic apoptosis can be induced in lymphoid cells, with impact on cytokine
production and parasite evasion from immune responses. Macrophages ingesting apoptotic leukocytes become
deactivated, as secretion of the pro-inflammatory cytokine TNF is suppressed by autocrine and paracrine secretion of
PGE2 and TGF [103]. Therefore, the phagocytic burial of apoptotic cells could play a detrimental role by deactivating
host macrophages, thus facilitating intracellular replication of parasites.
Moreover, injection of apoptotic cells increases the parasitemia of T. cruzi infected mice. Freire-de-Lima et al., [104]
have identified a biochemical pathway initiated in macrophages by contact with apoptotic cells and engagement of V3
integrin. This interaction stimulates PGE2 and TGF production, followed by increased ODC activity, and increased
synthesis of the polyamine putrescine [104, 105].
   Previous studies demonstrated that the intracellular pathogens are able to influence the production of various
effectors molecules by disabling intracellular signaling pathways required for their synthesis [106- 108].
   During T. cruzi infection spleen macrophages from BALB/c mice secreted lower concentration of nitrites than
C57BL/6 mouse cells, indicating that impaired function of myeloid cells in susceptible mice could contribute to the
persistence of infection [109]. In contrast to the Type 2 response seen in BALB/c, when C57BL/6 mice are immunized
with Cz, the induced cytokine profile was predominantly type 1 [110] indicating that the immunization of C57BL/6
mice with Cz primed the spleen cells for classical macrophage activation. Thus, it seems clear that classical activation
of macrophages by inflammatory cytokines plays a central role in the control of parasitemia and clearance during acute
phase of T. cruzi infection; however the alternative activation favors the replication, persistence and dissemination of
the parasite. Recently, in a comparative study during acute T. cruzi infection, it has been demonstrated that hepatic
leukocytes from infected B6 mice produced higher amounts of inflammatory cytokines than susceptible BALB/c mice,
whereas the anti-inflammatory cytokines IL-10 and TGFβ were only released by hepatic leukocytes from BALB/c.
Strikingly, a higher expression of TLR2 and TLR4 was observed in hepatocytes of infected BALB/c mice. However, in
infected B6 mice, the strong pro-inflammatory response was associated with a high and sustained expression of TLR9
and iNOS in leukocytes and hepatic tissue respectively. Additionally, in infected B6 mice, co-expression of gp91- and
p47- phox NADPH oxidase subunits were detected in liver tissue of infected B6 mice. Notably, the pre-treatment
previous to infection with Pam3CSK4, TLR2-agonist, induced a significant reduction of transaminase activity levels
and inflammatory foci number in livers of infected B6 mice. Thus, an altered TLR2, TLR4 and TLR9 signaling and
exacerbate inflammatory cytokine profile could be responsible of the fatal hepatic damage observed in infected B6.
These results demonstrate the importance of adequate TLR signaling on immune and non- immune cells to attenuate the
tissue damage during Trypanosoma cruzi acute infection [76, 111].
   It has been postulated that T. cruzi molecules, such as glycophosphatidylinositol anchors, may directly initiate IL-12
and NO production, through TLR2 activation, thus promoting host resistance during infection [95, 96]. However, it was
also demonstrated that Glycoinositolphospholipids from T. cruzi (GIPL) interfere with macrophages and dendritic cell
responses by down regulation of pro-inflammatory cytokines and co-stimulatory markers. Brodskyn C. et al.; 2002
showed that the ceramide portion of GIPL was responsible for most of the activity exhibited by the whole molecule.
Considering the important role of the immune response in determining the fate of the host-parasite relationship, the
immunoregulatory activities of T. cruzi GIPL are potentially important for parasite evasion and then pathogenesis of
infection with protozoan parasites [112]. Innate immunity plays a crucial role to efficiently develop the adaptive
immune response. In this context it was freshly reported that insufficient TLR activation contributes to the slow
development of CD8+ T cell responses in T. cruzi infection. The authors propose that T. cruzi is a stealth invader,
largely avoiding recognition by components of the innate immune system until the infection is well established. This
conclusion is supported by the ability to accelerate the induction of T cell responses to T. cruzi by administration of
ligands for TLR2 and TLR9 at the time of infection. These studies highlight a previously unappreciated mechanism of
immune evasion, the surreptitious establishment of infection, by the protozoan T. cruzi [113].


         5. Concluding remarks
The presented data highlight the fact that ROS and RNS are involved in the control of T. cruzi infection. However, the
impact of the microbicidal status achieved by phagocytes is not independent of other immune events, such as the levels
of inflammatory cytokines as well as second activation signals as PAMPs. Thus, the protective immune response
against microorganisms is multifactorial, and NADPH oxidase, ROS, iNOS, NO, and RNS have been significantly
implicated. It is noteworthy that during the very early stage of T. cruzi infection the interaction between host
macrophage and parasite itself or parasite derived antigens results in the induction of intracellular signaling pathways
which also lead to activation or deactivation of the cellular microbicidal mechanisms , which may either inhibit parasite
replication or provide a favorable environment to its dissemination. For this reason is particularly important to elucidate
the mechanism involved in the defense against pathogens as well as the evasive counterpart, in order to develop
successful strategies against infectious diseases.

Acknowledgements       Financial support received from Consejo Nacional de Investigaciones Científicas y Tecnológicas
(CONICET), Agencia Nacional de Investigaciones Científicas y Técnicas, and Universidad Nacional del Comahue is gratefully
acknowledged. S. Gea and N Guiñazú are members of Research Career from CONICET.


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