Besides parasite-derived factors playing a role in resistance to normal human serum (NHS), some parasite-derived molecules (see **Figure 1**, blue panel) are also able to dampen pro-inflammatory responses (TNF, NO) by classically activated macrophages (M1), needed for initial parasite control. One such important *T. brucei* protein is the Kinesin Heavy Chain 1 (TbKHC1) (87), which is released by the parasites in the environment via an unknown mechanism and sustains the development of the first (most prominent) peak of parasitemia in the blood and its control by the host. Following binding of TbKHC1 to the SIGN-R1 molecule (i.e., a surface C-type lectin expressed mainly by marginal zone macrophages within the spleen), the arginine/ NO metabolism is modulated in favor of arginase activity via an IL-10-dependent induction of arginase-1 and down-regulation of iNOS activities. In turn, this stimulates the production by the host of l-ornithine and hereby the synthesis of polyamines, which are essential nutrients for growth of trypanosomes in the host (88).  Another factor that trypanosomes use to establish infection comprises in the large family of transmembrane receptor-like adenylate cyclases (AdCs), called *T. brucei* Adenylate Cyclase (TbAdC) (89), which converts ATP into cyclic adenosine monophosphate (cAMP). During steady-state conditions, the TbAdC levels are low as is the cAMP production, yet upon stress (such as phagocytosis by M1 cells) the TbAdC levels can be elevated ~250-fold above the basal cellular content (89, 90). Subsequently, the cytoplasmic cAMP levels within the phagocytes increase, activating protein kinase A and leading to the inhibition of the synthesis of the trypanolytic cytokine TNF (91, 92). Hence, it seems that trypanosomes have developed a system whereby altruistic parasites are phagocytosed, thereby disabling the M1-mediated innate immune response required for parasite control (see **Figure 1**), and paving the way for initiation and establishment of the first wave of parasitemia.

#### Surface Coat Remodeling Prevents Elimination by the Humoral Immune Response

Given that AT are strictly extracellular parasites, they are confronted with the hosts' humoral immune response. Yet, one of the most fundamental changes occurring when parasites are inoculated into the mammalian host is the remodeling of the parasite cell surface (93). Indeed, within the mammalian host the metacyclic forms rapidly transform into the typical LS bloodstream forms expressing a different uniform VSG coat (94). This VSG coat consists of 5 × 106 homodimers of 50–60 kDa subunits held on the extracellular face of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (95), which consists of a ethanolaminephosphate-6-mannose-α1,2-mannose-α1,6 mannose-α1,4-glucosamine-α1,6-myo-inositol-1-phospholipid motif and a short galactose chain (96–99). Despite great variations in primary sequence, the secondary and tertiary structural features are highly conserved within the ordered coat structure (100). Although VSG molecules are free to diffuse in the plane of the membrane (101, 102), this ~15-nm-thick VSG coat has a dual role: shield off buried invariant proteins from recognition by the hosts' innate/acquired immune system and protect bloodstream parasites against complement-mediated lysis. Indeed, activation of the alternative pathway, which occurs in the absence of specific antibodies (Abs), may potentially play a crucial role in parasite clearance during the early stage of infection. Yet, it was shown by Devine et al. (103) that *T. b. gambiense* parasites, which are covered by C3, specifically inhibit the activation of the alternative pathway through their VSGs by masking sites on the plasma membrane, which are capable of promoting alternative pathway activation (104). Hence, the activation of the alternative pathway did not proceed further than the establishment of the C3 convertase, thereby impairing the generation of the terminal complex (C5–C9) which normally induces trypanolysis (103). In addition, soluble complement molecules, such as C3a and C5a, secreted during early stages of trypanosome infection, can further contribute to the initiation of the early inflammatory immune response within the chancre and may also act as (i) chemotactic agents attracting phagocytes to the site of infection and (ii) release histamine from mast cells thereby increasing microvascular permeability (105), which would allow/enable parasite extravasation into the blood circulation. Of note, the classical pathway, activated by immune complexes of trypanosome antigens and Abs, seems to contribute to trypanosome clearance through antibody-mediated trypanolysis and/or phagocytosis, which is of importance during peak parasitemia clearance (see later). Yet, also in this scenario, parasites are able to eliminate/remove surface-bound IgG (immune complexes) as well as complement through their rapid VSG recycling system and thereby prevent elimination (106). Furthermore, since complement is essential in antibody-mediated destruction of trypanosomes, by releasing vast amounts of soluble VSG (sVSG), mainly observed at the peak of parasitemia, this will scavenge complement factors and, hence, induce a state of hypocomplementemia (107, 108). This might favor the survival and escape of a minority of the parasites.

Additionally, binding of anti-VSG IgG or IgM to the trypanosome's coat results in parasite aggregation. Yet, trypanosomes are able to disaggregate in an energy-dependent manner involving protein kinase-C as part of the defense against the host humoral immune system (109). Hence, this could function as a survival strategy in the presence of antibody prior to the occurrence of VSG switching (109). The parasite's surface consisting of repetitive monotypic VSG molecules can cross-link B cell receptors (BCRs) and subsequently lead to T-cell-independent B-cell activation (110). However, during the process of antigenic variation (from metacyclic form toward trypomastigote form) mediated via changing VSG expression sites (i.e., *in situ* switching or transcriptional control) or by gene replacement resulting in a switch of the terminal telomeric VSG gene, heterologous VSG molecules are presented on the surface, thereby forming a mosaic VSG coat, which prevents direct B cell activation until a VSG uniformity is obtained (111, 112). This in turn might allow parasites to transiently escape T cell-independent B cell-mediated elimination and gives time to transform into trypomastigote forms adapted to survive in the mammalian host. Hence, this process gives the parasites an immunological advantage during the process of antigenic variation and is an efficient mechanism to escape antibody-mediated elimination during the early as well as chronic stage of infection (111).

#### VSG and VSG-Derived Fragments Trigger Different Cellular Innate Immune Responses

The VSG coat plays a key role in the interaction with the host, whereby it is involved in a population survival strategy through antigenic variation as well as in an individual cell survival strategy through rapid endocytosis, removal of bound antibody, and recycling back to the cell surface (106). The parasites not only use the VSG as an efficient escape mechanism jeopardizing the induction of an effective antibody response (113–115), but also use it as means to modulate the hosts' cellular responses. Indeed, *T. brucei* parasites contain an endogenous phospholipase C (PLC) known as the GPI-PLC, which is activated upon hypotonic lysis, stress, or during antigenic variation (90, 116, 117), and shown not to be essential but rather to act as a virulence factor given that a PLC−/− mutant was attenuated in mice (118). Activation of the GPI-PLC hydrolyzes the GPI-anchor on the VSG (119, 120). This hydrolysis will convert the hydrophobic membrane-form VSG (mfVSG) into a water sVSG (117), thereby leaving the dimyristoyl glycerol (DMG) compound of the GPI-anchor in the membrane and releasing the glycosylinositolphosphate (GIP)-VSG part (121). Both components (DMG and GIP-VSG) exhibit distinct functions as far as activating potential of host immune cells is concerned (121, 122). Indeed, the GIP-VSG moiety is recognized by a Type A scavenger receptor expressed on myeloid cells, thereby initiating the activation of NF-κB and MAPK pathways and the expression of pro-inflammatory genes, such as TNF-α, IL-6, IL-12p40, and GM-CSF (123). This is further amplified when myeloid cells are primed with T-cell derived IFN-γ (124). Hereby, the galactose side chain of VSG is responsible for TNF-α production following activation of the protein tyrosine kinase (PTK) pathway (121, 125). However, reversing the order of exposure (i.e., exposing myeloid cells to GIP-VSG before IFN-γ stimulation) resulted in a downregulation of IFN-γ-inducible responses, including transcription of inducible NO synthase and secretion of NO, which was associated with reduction in the level of STAT1 phosphorylation (126). This event might be of importance during the initial stage of infection, i.e., when sVSG is released from metacyclic forms during the early transition into bloodstream forms (see **Figure 2**). The GPI moiety, and in particular its DMG anchor that is released mainly during the descending phase of acute infection and during chronic infection, activates the protein kinase-C (PKC) pathway, and mediates macrophage priming/hyperactivation and LPS hyper-responsiveness in a MyD88-dependent manner (121, 122, 127, 128). Importantly, also in experimental bovine models, the DMG compound was shown to be crucial for M1 over-activation (129). In addition, the DMG compound of the mfVSG anchor seems to be crucial, via its IL-1α-inducing and -priming activity, in further fueling TNF induction (130).

#### CpG DNA is Used as Immunomodulatory Molecule to Trigger Macrophage Activation and Early Polyclonal B Cell Activation

Non-mammalian genomic DNA (i.e., CpG DNA) can also induce a host immune response (131). In this context, low amounts of tsetse-inoculated metacyclic parasites or SS blood-stream form parasites, continuously generated during the parasite cycle in the mammalian host, are eliminated/lysed giving rise to release of CpG DNA into circulation (see **Figure 2**). In turn, these CpG oligonucleotides trigger TLR9 signaling leading to the induction of M1 activation and polyclonal B-cell activation and subsequent isotype switching (132, 133). Importantly, CpG-mediated signaling can independently or synergistically with parasite-derived lipid or protein molecules (see further) activate the production of pro-inflammatory cytokines and NO needed for optimal peak parasitemia control. Indeed, as shown by Drennan et al. (127), during *T. brucei* infections, there is partial requirement for TLR9 signaling in the production of IFN-γ and VSG-specific IgG2a antibodies and for mammalian TLR family and MyD88 signaling in the innate immune recognition of *T. brucei*. Polyclonal B-cell activation, on the other hand, which is induced independently of BCR specificity, may play an important role in the defense against infections by enhancing natural antibody production and inducing memory B cells. Hence, polyclonal B cell activation increases the levels of natural antibodies to keep up with multiplication of the microorganisms, thus containing pathogen dissemination. Although triggering of polyclonal B cell activation is a natural innate immune response induced by many pathogens, the induction of polyclonal B cell activation (B cell expansion) might also be used as an immune evasion mechanism, whereby unselectively differentiating B cells can differentiate into short-lived plasmablasts (producing unspecific IgM), which ultimately results in apoptosis/elimination of the targeted B cell population (134, 135). In addition, regulatory B cells might also be induced and exert an immunosuppressive function by the secretion of IL-10, IL-17, IL-35, and transforming growth factor-β (TGFβ), and thereby dampen the initial pro-inflammatory immune response aimed at controlling infection (110, 136). However, so far no evidence of the occurrence of regulatory B cells is provided in this model.

#### TLTF Triggers IFN-**γ** Production by CD8**+** T Cells

Another trypanosome-derived factor documented to play a key role in early parasite–host interactions is the trypanosomederived lymphocyte-triggering factor (TLTF), a secreted 185 kDa invariant glycoprotein able to trigger IFN-γ production by CD8+ T cells (137–140). It was shown by Hamadien et al. (138) that early during *T. brucei* infection (day 3 p.i.) high levels of TLTF could be measured in the serum prior to IFN-γ production. Yet, later on during infection, these levels declined and coincided with increased levels of anti-TLTF antibodies. Of note, it was suggested that detection of TLTF and anti-TLTF antibodies in cerebrospinal fluid of HAT patients could be used as a tool for detection and staging of the disease (141). In addition, work from the same group and Nishimura et al. (142), revealed that IFN-γ was also able to trigger TLTF secretion in *in vitro* cultures of *T. brucei* parasites in a dose and tyrosine protein kinase-dependent manner and to stimulate parasite growth (143, 144). This suggests that TLTF and IFN-γ exert bidirectional activating signals between parasites and CD8+ cells. Hence, these molecules might play a crucial regulatory function in the parasite–host interactions and influence the disease course during experimental African trypanosomosis (see **Figure 2**), whereby (i) TLTF released by *T. brucei* parasites triggers early IFN-γ production by CD8+ T cells leading to the activation of M1 cells, (ii) IFN-γ triggers further secretion of TLTF by the proliferating parasites and was also suggested to be a growth factor for trypanosomes (142, 145). However, an alternative explanation for the apparent IFN-γ-mediated parasite growth effects, which cannot be excluded in *in vivo* settings, is that the early expansion of proliferating parasites (cf. ascending phase of first peak parasitemia) releases more TbKHC which in turn stimulates the synthesis of the essential nutrients, i.e., polyamines (see above) (88).

Figure 2 | Trypanosome establishment within the mammalian host. Within the blood circulation (several days post infection) the metacyclic trypanosomes give rise to a first small peak (which is not always observed). Subsequently, the metacyclic trypanosomes change their metacyclic VSG coat into the bloodstream VSG, thereby expressing a mosaic VSG that prevents Ab-mediated elimination. This dense VSG coat also prevents recognition of buried epitopes, including binding of complement factors (C3) to their surface. Also Ab-mediated elimination is prevented due to the rapid recycling of these VSG–Ab complexes and VSG shedding (i.e., sVSG release) that in turn scavenges circulating complement. Recognition of sVSG via SR-A on myeloid cells, in concert with CpG recognized via TLR9, results in the activation of MPC, which trigger activation of NK/NKT and T cells. In turn, these cells produce IFN-γ needed for proper activation of myeloid cells (M1 cells) and subsequent release of pro-inflammatory mediators (TNF/NO). Of note, initially, when GIP-sVSG is released via PLC activation due to stress prior to IFN-γ production, there is a weak activation of myeloid cells. Yet, triggering of PRR at the level of B cells (i.e., TLR9 via CpG) can also lead to polyclonal B-cell activation. Subsequently, parasites rapidly multiply as LS forms giving rise to the most prominent parasitemia peak. Trypanosomes also release TLTF that triggers IFN-γ production by CD8+ T cells, which in turn stimulates parasite proliferation. However, IFN-γ exposure in concert with GIP-sVSG release will trigger an enhanced production of trypanolytic molecules by myeloid cells, which in concert with anti-VSG antibodies are needed for peak parasitemia control. Upon reaching the peak of parasitemia, the majority of the parasites differentiate into non-proliferative SS forms that are pre-adapted for uptake by tsetse flies, while a minority undergoes antigenic variation. Yet, in the mammalian host, these SS forms are deemed to die, thereby releasing mfVSG as well as CpG. These molecules exert dual functions; (i) the DMG of mfVSG triggers macrophage hyperactivation and LPS-hypersensitivity, while CpG further fuels polyclonal B-cell activation. These B cells can differentiate into short-lived plasmablasts (producing unspecific IgM) and ultimately results in apoptosis/elimination of all B-cell subsets and loss of B-cell memory. At this stage of infection, parasites also release TSIF that further stimulates the production of suppressive M1 and induces T-cell suppression. Once the first peak of parasitemia is controlled, the infection is established and the hosts' adaptive immune response will develop, whereby the B- and T-cell response are impaired and there is a polarized M1 activation leading the trypanosomosis-associated pathogenicity.

#### TbTSIF Induces M1 Cells and Triggers T-Cell Suppression

Several trypanosome components have been shown to exert a macrophage-activating potential, leading to NO-dependent suppression of T-cell proliferation (146, 147). Another parasite-derived molecule exerting the potential to modulate the host immune network is the *T. brucei*-derived Trypanosome Suppression Immunomodulating Factor (TSIF) (148). Since this molecule plays a role in triggering suppressive M1, it is most likely released during the descending phase of infection (see **Figure 2**), at the moment that M1 cells exert their most prominent effects (i.e., production of trypanolytic molecules TNF/NO). Furthermore, as shown by Gomez-Rodriguez et al. (148), this molecule is able to (i) block T-cell proliferation in a cell–cell contact and IFN-γ/NO-dependent manner and (ii) limit secretion of immune-protective IL-10 by alternatively activated macrophages (M2) required to dampen M1-mediated pathogenic effects. Hence, TbTSIF could play a dual role, i.e., contribute to initial parasite control (via TNF/NO) and fuel suppressive M1 and T-cell suppression leading to pathogenicity. However, T-cell suppression could also be a means of the parasite to negatively affect/inhibit B-cell development and thereby impair effective humoral responses (see later) and allow/guarantee parasite survival. In addition, it seems that TbTSIF is also essential for *T. brucei* development/biology since TbTSIF knock-out parasites were not viable and died within 2 days.

### Host Innate/Adaptive Responses Determine Trypanosome-Associated Pathogenicity

Since AT can establish chronic infections in their mammalian host, which is associated with different forms of pathogenicity (anemia, liver injury, weight loss, neuropathology,…), it is clear that the innate response is insufficient for complete elimination of the parasites and, hence, will require the help of the adaptive immune response to combat infection. Yet, the modulation of the innate immune response might also affect the rejoinder of the adaptive immune response. In this section, we will elaborate on what is happening during the later stage of infection once the trypanosome infection is established in the mammalian host.

#### Trypanosome-Infections Impair B-Cell Functionality

As mentioned before, although trypanosomes use antigenic variation of their VSG coat as an efficient way to escape host humoral responses, trypanosomes also directly/indirectly affect B cell development as an additional means to escape elimination. Important to mention is that experiments in μMT (B cell deficient) and IgM−/− mice revealed that the initial development of peak parasitemia is independent of infection-induced anti-VSG antibodies. In addition, *in vivo* parasite VSG switching is an intrinsically programed genetic process that is independent of B cells or antibody pressure, with the function of antibodies mainly limited to the elimination of the remaining non-switched parasites (149). Studies in experimental rodent infection models have implicated T-cell-independent anti-VSG IgM responses to be the first line of host defense against proliferating parasites (150) (see before). Although B cells aid in periodically clearing circulating parasite levels by VSG-specific antibodies, they are limited by their VSG-specificity, yet they are required for longterm survival, while IgM antibodies play only a limited role in this process (149, 151–153). Importantly, similar observations were obtained in a Cape Buffalo model for natural trypanosomosis resistance (154). An additional aspect that plays a role in antibody-mediated recognition of trypanosomes is that though polyclonal antibodies are raised against different parts of the VSG molecule (155), only surface exposed regions (N-terminal more variable region) of the VSG could play a role in parasite elimination given that the buried epitopes (C-terminals more conserved region) are inaccessible for conventional antibodies (102, 156). Indeed, the VSG coat functions as a protective coat shielding of conserved buried epitopes/proteins, thereby preventing elimination of successive waves of trypanosomes expressing a different VSG coat.

The data so far indicate that *T. brucei* parasites affect B cells already early during infection (within 1 week p.i.) at different levels, resulting in the loss of humoral immune competence in trypanosusceptible hosts. This early undermining of humoral responses is important given that the production of high-affinity, antigen-specific, class-switched, antibodies takes up to 10 days after immunization (157). First, as mentioned before, there is induction of non-specific, polyclonal B-cell activation leading to clonal exhaustion (158–160). Second, there is destruction of the splenic B cell compartment that is manifested by the occurrence of marginal zone and follicular B cell (FoB) depletion. Hereby, IFNγ was shown to play a key role in destruction of FoBs (161), which was associated with enhanced expression of the death receptor Fas, leading to loss of protective B cell memory responses against unrelated antigens. Third, it was shown that during *T. brucei* infection there is an impaired B-cell lymphopoiesis in the bone marrow and spleen already at the level of transitional B cells (159, 162). Hereby, there was massive cell death observed in transitional B cells *in vitro* through a contact-dependent mechanism, which is not dependent on TNF or prostaglandin-dependent death pathways (159). Of note, the mechanism(s) of *T. brucei*-induced transitional B-cell depletion *in vivo* remains to be fully elucidated.

Collectively, trypanosomes deliberately undermine the host's capacity to sustain antibody responses against recurring parasitemia waves by depleting transitional B cells, which in turn impairs the replenishment of the mature marginal zone and FoB populations. Since parasite-specific antibodies are essential for parasite control, inhibition of B-cell maturation at the transitional stage is an efficient evasive mechanism to prevent the buildup of protective "humoral" immunity against successive parasitemia waves. In this context, it was recently shown by De Trez et al. (163) that *T. brucei* infection is impairing the maintenance of the antigen-specific plasma B-cell pool.

#### Trypanosome Infections Induce Early IFN-**γ**-Mediated M1 Polarization that Subsequently Contributes to Pathogenicity Development

The parasite-derived components sVSG and CpG DNA that are released trigger via specific receptors (SR-A, TLR9) myeloid cell activation (121–123, 127, 164). In turn, this triggers T cell activation and the release of IFN-γ (165), which primes macrophages to become fully activated/M1 polarized thereby releasing pro-inflammatory molecules (TNF/NO) needed for parasite control (166, 167). This type 1 cytokine storm can also culminate in pathology development if maintained during later stages of infection (166– 172). Yet, only animals able to produce tissue-protective IL-10 can exhibit an alleviated pathogenicity (167). Importantly, the balance of these different activation/deactivation signals may determine the outcome of infection (173, 174). Recently, it was shown that different lymphocyte populations play a role in IFN-γ production, whereby NK and NKT cells are the earliest IFN-γ producers, followed by CD8+ and CD4+ T cells (124). A possible explanation for this transition in different IFN-γ-producing T cells during the early stages of infection could be that: (i) initially type-I IFN released by for instance TLR9-activated myeloid cells can trigger NK/NKT-cell activation (175, 176); (ii) subsequently, parasite-derived TLTF will trigger IFN-γ production by CD8+ T cells in a non-antigen-specific manner (140, 145, 177); and (iii) finally, the increased release and subsequent processing of sVSG will lead to MHC-II presentation and activation of CD4+ T cells, thereby further fueling IFN-γ production and M1 polarization (178).

Whatever the source of IFN-γ may be, research so far indicates that early IFN-γ production triggers an acute inflammatory reaction resulting in acute anemia development, as witnessed by a 50% reduction in circulating red blood cells (RBC) within 2 days following peak parasitemia. After a short recovery phase, a subsequent gradually increasing loss of RBCs occurs during the chronic infection stage (166, 167, 179). Of note, anemia development was found to be independent of antibodies and the height of the parasitemia peak, whereby the acute nature of this phenomenon implies a consumptive etiology (149, 168). IFN-γ plays also a crucial role in the recruitment and activation of erythrophagocytic myeloid cells. In addition, the work of Cnops et al. (124) indicates that the absence of NK, NKT, and CD8+ T cells, but not CD4+ T cells, during the early stage of infection results in a reduced anemic phenotype similar to IFNγR−/− mice. In addition, it was recently shown that trypanosomes can release extracellular vesicles (EV) that can fuse to mammalian erythrocytes thereby changing their physical properties and making them more susceptible to erythrophagocytosis (86). This in turn leads to acute anemia and could be a means of the parasites to acquire essential nutrients [hemoglobin and/ or iron (see before)]. Hence, both host-induced and parasiteinduced factors could account for acute anemia development. Subsequently, the hosts' ability to respond to the acute anemia will determine whether anemia persists or not during the chronic phase of infection (55, 166, 180–182).

Another pathological feature associated with *T. brucei* infections is neuropathology, whereby parasites pass the BBB and cause severe neurological complications. Interestingly, the work of Amin et al. (183) showed that *T. b. brucei* parasites penetrate the BBB very early during infection (within 2–3 days post infection), whereby they proposed that TLR9 and MyD88-mediated activation of DCs triggers via type-I IFN (IFN-α/β) T-cell activation. Subsequently, these activated T cells invade the central nervous system (CNS) in a IFN-α/β, IFN-γ and TNF-dependent manner, whereby TNF can induce the expression of adhesion molecules (ICAM-1 and VCAM-1) in brain endothelial cells in a TNFR1-dependent manner and contributes to the leakiness of inter-endothelial cell tight junctions or stimulation of matrix metalloproteases activities that open the parenchymal basement membranes (184, 185). Furthermore, the same group showed that IFN-γ, as well as the IFN-inducible chemokine CXCL10, promotes the penetration of T cells and parasites in the brain (186, 187), suggesting that parasites can also follow T cells during their brain invasion across the BBB. However, the work of Frevert et al. (188) showed, using a murine model and intravital brain imaging, that bloodstream forms of *T. b. brucei* and *T. b. rhodesiense* enter the brain parenchyma within hours post injection, before a significant level of microvascular inflammation is detectable. Yet, there are differences in the trypanosome strain used and the infection dose as well as the route of infection that

### REFERENCES

could account for the different results. Collectively, it seems that whatever mechanism (host-mediated or not) parasites use to pass the BBB and infiltrate the brain, extravasations of parasites from the blood into the brain might be an alternative evasion mechanism to escape humoral responses that predominate in the blood circulation and thereby allow future transmission when parasites migrate back into the blood.

## CONCLUSION AND PERSPECTIVES

Overall, it seems that trypanosomes have evolved efficient immune escape mechanisms to sculpture the hosts' innate/adaptive immune response in order to establish an environment suitable for parasite survival and transmission. This manipulation of the host response has its cost since this undermines the hosts' capacity to respond/recover following establishment of the parasites. Hereby, persistence of inflammation during the chronic stage of infection culminates into pathogenicity and subsequent death if left untreated. Hence, identification of host-derived factors playing a role in persistence of inflammation could be an alternative means to alleviate trypanosomosis-associated pathogenicity. In this context, it was recently shown that the pleiotropic host molecule macrophage migration inhibitory factor (MIF) plays a key role persistence of inflammation and infection-associated pathogenicity (180). Hence, future intervention strategies against African trypanosomosis might require a dual approach, i.e., development of efficient anti-trypanosomal agents combined with neutralization of anti-pathogenicity inducing "host" factors, which combined might allow reducing the economical losses of the affected continents.

### AUTHOR CONTRIBUTIONS

BS, GC, JA, JG, SM, and CT wrote the manuscript.

### FUNDING

This work, performed in frame of an Interuniversity Attraction Pole Program (PAI-IAP N. P7/41, http://www.belspo.be/belspo/ iap/index\_en.stm), was supported by grants from the FWO (KaN 1515813N and G031312N), an EU/FP7 ERC starting grant (No.282312), an UA research fund (TT-ZAPBOF 33049) and the Strategic Research Program (VUB). BS was supported by the Strategic Research Program (SRP3, VUB). The authors declare that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Stijlemans, Caljon, Van Den Abbeele, Van Ginderachter, Magez and De Trez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Malaria-Cutaneous Leishmaniasis Co-infection: Influence on Disease Outcomes and Immune Response

Raquel A. Pinna1 , Danielle Silva-dos-Santos2 , Daiana S. Perce-da-Silva1 , Joseli Oliveira-Ferreira3 , Dea M. S. Villa-Verde2 , Paula M. De Luca3 \* and Dalma M. Banic1 \*

1 Laboratory of Simulids, Onchocerciasis and Sympatric Diseases: Mansonelliasis and Malaria, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, 2 Laboratory of Thymus Research, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, 3 Laboratory of Immunoparasitology Research, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil

Malaria and Cutaneous Leishmaniasis (CL) are co-endemic throughout large regions

#### Edited by:

Emilio Luis Malchiodi, University of Buenos Aires, Argentina

#### Reviewed by:

Hira Nakhasi, United States Food and Drug Administration, USA Rodolfo Cordeiro Giunchetti, Universidade Federal de Minas Gerais, Brazil

#### \*Correspondence:

Paula M. De Luca<EMAIL_ADDRESS>Dalma M. Banic<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology

Received: 25 March 2016 Accepted: 07 June 2016 Published: 27 June 2016

#### Citation:

Pinna RA, Silva-dos-Santos D, Perce-da-Silva DS, Oliveira-Ferreira J, Villa-Verde DMS, De Luca PM and Banic DM (2016) Malaria-Cutaneous Leishmaniasis Co-infection: Influence on Disease Outcomes and Immune Response. Front. Microbiol. 7:982. doi: 10.3389/fmicb.2016.00982 in tropical countries and co-infection may impact the evolution of host-parasite interactions. In the present study, we evaluate Malaria/Leishmaniasis disease outcome, Th1/Th2 cytokine levels and the CD4 and CD8 T-cell profiles in a co-infection murine model (BALB/c) of Plasmodium yoelii 17XNL (Py) and Leishmania amazonensis (La) or L. braziliensis (Lb). Malaria parasitaemia was assessed through blood strains stained with Giemsa. Leishmania lesions were monitored with a digital caliper and parasite loads determined by limiting-dilution assay. Serum levels of IFN-γ, TNF, IL-2, IL-4, IL-6, IL-10, and IL-17 were determined using multiplexed bead assay and expression of CD3, CD4, and CD8 T-cells markers were determined by Flow Cytometry in the thymus, spleens and lymph nodes. Parasitaemia in Lb+Py co-infected group was lower than in Py singleinfected group, suggesting a protective effect of Lb co-infection in Malaria progression. In contrast, La+Py co-infection increased parasitaemia, patent infection and induced mortality in non-lethal Malaria infection. Regarding Leishmaniasis, Lb+Py co-infected group presented smaller lesions and less ulceration than Lb single-infected animals. In contrast, La+Py co-infected group presented only a transitory delay on the development of lesions when compared to La single-infected mice. Decreased levels of IFN-γ, TNF, IL-6, and IL-10 were observed in the serum of co-infected groups, demonstrating a modulation of Malaria immune response by Leishmania co-infections. We observed an intense thymic atrophy in Py single-infected and co-infected groups, which recovered earlier in co-infected animals. The CD4 and CD8 T cell profiles in thymus, spleens and lymph nodes did not differ between Py single and co-infected groups, except for a decrease in CD4+CD8+ T cells which also increased faster in co-infected mice. Our results suggest that Py and Leishmania co-infection may change disease outcome. Interestingly Malaria outcome can be altered according to the Leishmania specie involved. Alternatively Malaria infection reduced the severity or delayed the onset of leishmanial lesions. These alterations in Malaria and CL development seem to be closely related with changes in the immune response as demonstrated by alteration in serum cytokine levels and thymus/spleens T cell phenotypes dynamics during infection.

Keywords: co-infection, Malaria, Leishmaniasis, immunology, cytokine, Plasmodium yoelii, Leishmania braziliensis, Leishmania amazonensis

### INTRODUCTION

fmicb-07-00982 June 24, 2016 Time: 16:57 # 2

Malaria and Cutaneous Leishmaniasis (CL) are two of the world's most important vector-borne parasitic diseases (World Health Organization, 2010, 2015; Alvar et al., 2012). Malaria, an infectious disease caused by Plasmodium genus parasites, is an important cause of global mortality and morbidity. Half of the world population is at risk of contracting Malaria, with approximately 214 million cases and 438 000 deaths in 2015, amongst the 3.2 billion people living at risk of infection (World Health Organization, 2015). Humans can be infected by five Plasmodium species: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Although P. falciparum accounts for the great majority of morbidity and mortality, P. vivax has a wider geographic distribution and causes considerable symptomatic disease (Battle et al., 2014). Malaria infection has a variable clinical phenotype, ranging from a mild febrile illness to severe disease and death, but infection can also occur in the absence of clinical symptoms. These variations in disease pattern are attributable to many factors, including the genetic background of the host and pathogen, the complex relationship between the parasite and host immune response, the dynamics of parasite transmission and/or the biological interactions of the parasites within the host (Good and Doolan, 2010; van den Bogaart et al., 2012). Leishmaniasis is a complex disease caused by different species of intracellular protozoan parasites from the genus Leishmania, which also induces significant morbidity and mortality throughout the world. According to the World Health Organization (2016) 350 million people in 98 countries are at risk of infection. There are an estimated annual 1.3 million new cases worldwide, of which 300,000 cases are of Visceral Leishmaniasis (VL) and another 1 million of CL. Seventy (70) to 75% of the CL cases occur in Afghanistan, Algeria, Brazil, Colombia, Costa Rica, Ethiopia, Iran, Peru, Sudan, and Syria (World Health Organization, 2016). CL is caused by L. major, L. tropica, and L. aethiopica in the Old World, whereas in the New World it is most frequently caused by L. mexicana, L. amazonensis, L. braziliensis, L. panamensis, and L. guyanensis (World Health Organization, 2010; Hartley et al., 2014). Symptoms range from the more prevalent single selfhealing cutaneous lesions to uncontrolled parasite replication, producing non-healing cutaneous, mucosal or even visceral disease as well as chronic metastatic dissemination throughout the skin. This spectrum of manifestations is multifactorial and depends on complex interactions among parasite, host, and environmental factors, including the Leishmania specie, genetic background and immunological status of the host (Hartley et al., 2014).

The overlapping geographic distribution of Malaria and Leishmaniasis, especially in the tropical and subtropical countries demonstrate clearly that the potential for interaction among these parasites may occur and play a role in determining disease outcome (Hotez et al., 2006; van den Bogaart et al., 2012, 2014). Despite this natural coexistence, data from concomitant infections are so far not available in the literature (Ab Rahman and Abdullah, 2011; van den Bogaart et al., 2012, 2014). Therefore, the impact of the dual infections on the human population health remains unassessed particularly in what concerns CL.

In the eighties, two studies in the murine model evaluated the effect of non-lethal P. yoelii and L. amazonensis concomitant infections in the course of each disease (Coleman et al., 1988a,b). These studies demonstrated that severity and susceptibility to both diseases were enhanced during co-infection. Since these parasites do not compete for the same host cells, neither anatomical sites nor resources, the interaction among them is most likely indirect and related mostly to the host's immune responses induced by each pathogen. However, in both studies (Coleman et al., 1988a,b) the immunological mechanisms involved were not investigated. Alternatively, several studies have shown that outcome of Malaria and CL is determined, in part, by the balance of pro- and anti-inflammatory immune responses (Louis et al., 1998; Li et al., 2001; Silveira et al., 2009; Taylor-Robinson, 2010; Medina et al., 2011; Freitas do Rosario and Langhorne, 2012; Kedzierski and Evans, 2014).

The immune response against the blood stages of Malaria parasites operate in concert and sequentially to control and clear the parasitaemia by an early and strong pro inflammatory, type 1 response (Th1), limiting parasite growth, followed by a shift to anti-inflammatory, type 2 immune (Th2) response. The balance between cytokines produced by pro inflammatory and anti-inflammatory responses during different phases of the blood stage infection determines the outcome of the disease (Li et al., 2001). In contrast to the strong type 1 immune response at the beginning of the Malaria infection, CL caused by L. amazonensis generally results in type 2 cellular immune response polarization (Silveira et al., 2009; Gollob et al., 2014). On the other hand, in CL caused by L. braziliensis, there is evidence that increased production of inflammatory cytokines (IFN-γ and TNF-α) and absence of IL-10 is associated with tissue destruction and the development of mucosal lesions (Dutra et al., 2011; Gollob et al., 2014; Oliveira et al., 2014). In this context, the current study was designed to evaluate the outcome of Malaria-CL infections, the CD4 and CD8 T-cell profiles as well as the Th1/Th2 cytokine levels in a co-infection murine model (BALB/c) of P. yoelii 17XNL (non-lethal) and L. amazonensis or L. braziliensis. Furthermore, as far as we know, this is the first report utilizing L. braziliensis and P. yoelii 17XNL non-lethal strain as a co-infection model.

#### MATERIALS AND METHODS

#### Animals, Parasites, and Infections

Female BALB/c mice (5–6 weeks old) were obtained from the Center for Laboratory Animal Breeding of the Oswaldo Cruz Foundation (FIOCRUZ) (Rio de Janeiro, RJ, Brazil). Mice were maintained under specific pathogen-free conditions in Experimental Animal Center of Leônidas Deane building (FIOCRUZ). This research protocol was approved by the Ethical Committee for Animal Use of FIOCRUZ/MS (license LW-17/11).

P. yoelii 17XNL (non-lethal strain) was provided by Dr Fábio T.M. Costa at Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, UNICAMP, Campinas, SP, Brazil. Parasite

stabilates were stored at −196◦C. To obtain the experimental inoculum of P. yoelii 17XNL, parasitized red blood cells (pRBCs) were defrosted and passed through three homologous donor mice.

Promastigotes of L. braziliensis (MCAN/BR/98/R69) and L. amazonensis (IFLA/BR/67/PH8), provided by Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, FIOCRUZ, RJ, Brazil, were cultured in Schneider's medium supplemented with antibiotics (200 IU penicillin and 200 µg streptomycin/ml) and 10% inactivated fetal calf serum (all from Sigma–Aldrich, St Louis, MO, USA). For L braziliensis 2% of sterile human urine was also added to the cultures (Howard et al., 1991).

Eight to nine-week-old mice (∼22g each) were divided randomly into six groups, **Figure 1**. Group C: comprised uninfected mice; Group Py: mice were injected intraperitoneally (i.p.) with 106 P. yoelii 17XNL pRBCs (0.2 mL); Groups Lb and La: mice were inoculated intradermally in both ears with either 105 or 104 stationary phase promastigotes of L. braziliensis or L. amazonensis, respectively, as previously described (Belkaid et al., 2000); Groups Lb+Py and La+Py: mice were coinfected with both P. yoelii 17XNL and either L. braziliensis or L. amazonensis. Each group had 25 to 35 mice and experiments were repeated at least three times. First, mice were infected with Leishmania sp. and 3 days later with P. yoelii 17XNL, as shown in **Figure 1**. At 5, 10, 17, and 25 days post-P. yoelii 17XNL infection, six animals from each group were bled, for serum cytokine assay, and euthanized, for thymus, spleen, ears and lymph nodes removal. Mortality of mice was monitored daily, post-P. yoelii 17XNL infection.

### Determination of Parasite Load, Leishmania Lesion Size, and Ulceration

Malaria parasitaemia was monitored every 2 days starting at day 3 post-infection. Blood smears were prepared from tail vein, methanol-fixed, stained with Giemsa and microscopically

examined to determine parasitaemia in 1,000 erythrocytes. The percentage of infected erythrocytes was calculated as follows: Parasitaemia (%) = (number of infected erythrocytes x 100)/total number of erythrocytes counted (1,000).

The diameter of dermal Leishmania sp. lesions were measured weekly with a digital calipter. We also monitored the time elapsed until ulcer formation in nodules. Parasite numbers in the ears and draining lymph nodes were determined as previously described (Belkaid et al., 2000) and scored as the highest dilution containing viable parasites after incubation for 6 days at 26◦C.

### Cytokines Quantitation in the Serum

The serum levels of cytokines (interferon – IFN-γ, tumor necrose factor – TNF, interleukin (IL)-2, IL-4, IL-6, IL-10, and IL-17) were determined using the BD Cytometric Bead Array Kit Mouse Th1/Th2/Th17 Cytokines (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Briefly, 25 µL of mouse serum was incubated for 2 h at room temperature with 25 µL of cytokine capture bead and 25 µL of phycoerythrin (PE) detection reagent. Then, samples were washed with 1 mL of buffer by centrifugation (200 × g, 5 min). The supernatants were carefully aspirated and discarded from each assay tube. Finally, beads were resuspended in 300 µL of buffer for analysis on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The serum cytokine concentrations (pg/mL) were determined using a standard curve from recombinant cytokines provided by the kit.

### Determination of Thymus Relative Weight

For determination of thymus relative weight, body and thymus of each mouse were weighted. Thymic index (TI) was calculated according to the following formula: TI = thymus weight (mg) / body weight (g).

### T Cell Phenotyping by Flow Cytometry

Thymus, spleens, mesenteric nodes, subcutaneous nodes (cervical, axillary, brachial, and inguinal) and submandibular nodes (draining ears lesions) were dissected and mechanically disaggregated. Single-cell suspensions from control, infected and co-infected mice were obtained in RPMI-1640 supplemented with 10% fetal calf serum (Gibco-Invitrogen, USA). Cell numbers and viability were determined by Trypan blue exclusion using Neubauer chamber.

For T lymphocytes phenotypic characterization, 106 cells were stained with FITC labeled anti-mouse CD4, APC labeled anti- mouse CD8 and PE labeled anti-mouse CD3 monoclonal antibodies. After incubation for 30 min, followed by 2 washes with staining buffer (PBS, 0,1% BSA, 0,01% sodium azide, all from Sigma–Aldrich), cells were fixed with 1% paraformaldehyde solution (Sigma–Aldrich), washed and resuspended in staining buffer until acquisition. A minimum of 30,000 events per sample were acquired inside the lymphocytes gate, based on size and granularity properties using a FACSCantoIITM flow cytometer (BD Bioscience, USA) and analyzed using FlowJo 7.5.5 software (Tree Star Inc. Ashland, OR, USA).

### Statistical Analysis

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Sample size was determined a priori using the software G\*Power version <IP_ADDRESS>, based on the Mann–Whitney test, a significance level of 0.05, and a minimum power of 80%. Results were evaluated by a non-parametric test, the Mann– Whitney test, using GraphPad Prism software version 5.0 (GraphPad Software, La Jolla, CA, USA). Data are expressed as means ± SEM and considered to be statistically significant when p < 0.05. Survival curves were analyzed by Mantel Cox and Gehan–Breslow–Wilcoxon tests and Leishmania lesions ulcerations were analyzed by Chi-square test using the same software.

### RESULTS

### L. braziliensis and L. amazonensis co-Infection exerts opposite effects on P. yoelii 17XNL Infection

Blood stage P. yoelii 17XNL parasites induced Malaria infection in all BALB/c mice from single-infected (Py) and L. braziliensis or L. amazonensis co-infected groups (Lb+Py and La+Py). Parasitaemia peak occurred between 10 and 17 days after infection and no parasite was detected after 23 days postinfection in blood smears of Py single-infected mice. The overall mean parasitaemia observed in Lb+Py co-infected group was lower than that observed in P. yoelii single-infected group (Py). Statistically significant differences observed in some days are suggestive for a protective effect of L. braziliensis coinfection in Malaria progression (**Figure 2A**). In contrast, co-infection with L. amazonensis appears to have a negative influence in acute Malaria (**Figure 2C**), and mortality in La+Py co-infected group enhanced in comparison to Py single infected group (**Figure 2D**). La+Py group exhibited higher parasitaemia on day 5 and a longer course of P. yoelii Malaria patency with mice remaining parasitemic 8 days longer than mice in Py single-infected group (**Figure 2C**). The higher rate of parasitemia in La+Py coinfeted mice seems to be associated with decreased survival. Four weeks after P. yoelii 17XNL infection, 30% of the co-infected La+Py mice were dead, compared to 100% of survival in the group infected only with P. yoelii 17XNL. Py single-infected groups showed no mortality during the whole experiment (**Figures 2B,D**).

### Co-Infection is Able to Reduce Severity of L. braziliensis Lesions

BALB/c mice were intradermally infected with 105 stationary phase promastigotes of L. braziliensis. Lb+Py co-infected group showed an overall tendency to present smaller lesion sizes than Lb single infected group (**Figure 3A**). In both groups, lesions reached maximum size 5 weeks post-infection and started to heal afterward. To evaluate disease severity we followed the onset of open ulcers in Leishmania-infected and co-infected groups (**Figure 3B**). Ulcers started to appear 21 days postinfection in Lb group and after 28 days in Lb+Py. At week 11 after infection (77 days) Lb single-infected group presented a higher number of ulcerated nodules (83.3%) than Lb+Py (40.9%). At week 17 and 18 post infection, there was a complete resolution of lesions in Lb+Py and Lb groups, respectively (data not shown). Limiting-dilution assays performed in the ears and in draining lymph nodes showed an earlier detection of parasites in the ears of Lb single-infected animals than in co-infected group (14 and 21 days post-infection, respectively) with no difference in parasite loads between groups. On the other hand, in the draining lymph nodes parasites were detected 28 days after infection in both groups, and parasite loads were lower in Lb+Py co-infected group (p = 0.006, **Figures 3C,D**).

### Co-Infection Reduce Severity of L. amazonensis Lesions Only during Acute Phase Malaria

Infection of BALB/c mice with 104 stationary phase promastigotes of L. amazonensis induced chronic lesions that did not heal over time in La and La+Py groups (**Figure 4A**). During murine Malaria (from day 0 to day 25) lesions in La+Py co-infected group were significantly smaller than in La single infected group. Although lesions of La group presented greater sizes than La+Py until 28 days of infection, after that time and until the end of the experiment both groups present similar lesion sizes. Ulcerated nodules were detected first in La group (21 days post-infection) while in La+Py group visual detection occurred 1 week later (28 days post-infection) (**Figure 4B**). However, after 63 days of infection all animals from both groups presented ulcerated nodules. Limiting dilution analysis performed at the site of infection and in draining lymph nodes showed higher parasite loads in La+Py group than La single-infected group (**Figures 4C,D**).

### Co-Infection Modulate Serum Cytokine Levels Induced by P. yoelii 17XNL

Cytokine levels were measured in serum samples obtained from 4 to 6 animals of each experimental group. As expected, increased levels of the proinflammatory cytokine IFN-γ, were observed in all groups infected with P. yoelii 17XNL (Py, Lb+Py, and La+Py) in the first week of infection (day 5). The levels of TNF, IL-6 and IL-10 also increased in the serum of the same three groups during blood stage Malaria. Cytokine levels in coinfected groups (Lb+Py and La+Py) were clearly lower than those observed in Malaria single infected group (Py) suggesting a modulatory effect of co-infection (**Figure 5**). We were not able to detect any of the analyzed cytokines in serum samples from control and Lb or La single infected animals, except for IL-10 in La infected group, only at day 25th post infection. Although we could observe a tendency for an increased production of TNF, IL-6 and IL-10 in the first 10 days of Malaria infection for Lb+Py co-infected group when compared to La+Py coinfected animals, statistic difference was observed only for IL-6 at day 10 post Py infection (P = 0.045). We were not able to detect the cytokines IL-2, IL-4, and IL-17 in serum

samples from all experimental groups, at any time of the study.

### Recovery of Thymic Atrophy Occurs Earlier in Co-Infected Animals

In the course of Malaria infection, we observed a significant reduction of the thymus relative weight in comparison to the uninfected control group. Thymic atrophy was first observed 10 days post P. yoelii infection (dpi) and persisted until day 25th of infection in Py single-infected group. In coinfected groups (Lb+Py and La+Py) thymus relative weight came back to normal values at 25 day post-infection and no significant difference could be observed in comparison to the control group. During the entire experiment, Leishmania single-infected groups showed no differences in thymus relative weight when compared do the uninfected control group (**Figure 6**).

Flow cytometry analysis showed that the thymus of Py and co-infected groups underwent a decrease in CD4+CD8+ doublepositive (DP) T cell subset and an increase in CD4+ singlepositive (SP), CD8+SP and CD4−CD8− double-negative (DN) T cell populations (**Figure 7**). A significant increase in CD4+SP thymocytes occurred at 17 dpi and persisted up to 25 dpi in Py single-infected animals. In Lb+Py group, CD4+SP thymocytes increased 10 days post infection, and returned to control values by day 25th post infection. In La+Py this transitory increase was observed latter at 17 dpi. The dynamics of CD8+SP thymocytes in single infected and co-infected groups exhibited the same profile of CD4+SP. When we evaluate this results together, it is possible to notice that in Lb+Py co-infection, the increase in CD4+SP and CD8+SP occurred sooner than in La+Py group (10

and 17 dpi, respectively). Double-negative thymocytes increased in Py single-infection at 17 and 25 dpi. The same increase occurred in co-infected groups only at day 17 post infection. In parallel, CD4+CD8+ DP cells significantly decreased in the thymus of Py single infected animals at 17 dpi and remained lower at 25 dpi. This decrease was observed earlier is Lb+Py group (10 dpi) but, at day 25th post infection, DP cells increased in this group and were higher than in control and single infected groups. Interestingly, in La+Py co-infected group, the increase in DP thymocytes occurred earlier (10 dpi) and was followed by a rapid decrease at day 17th post infection, coming back to control values 25 dpi. Overall, thymus recovery seems to

occur faster in Malaria/Leishmania co-infection than in Py single infection.

## Leishmania Co-Infection Delay the Enhancement of Double Negative Cells Induced by P. yoelii 17XNL Infection in the Spleen

We also evaluated the T cell phenotypes present in the spleens, mesenteric, subcutaneous and submandibular nodes (the last ones as the draining nodes from ears lesions) in single infected and co-infected animals. No alterations were observed in the T cell phenotypes present in subcutaneous and submandibular nodes among the studied groups, while in mesenteric nodes and spleens we were able to observe similar patterns of alterations that were more evident in the spleens (**Figure 8**). After a transitory increase at 10 dpi in Py single infected and in coinfected groups, CD4+ T cells dropped in Py single infected group at 17 dpi and remained bellow controls at 25 dpi. In Lb+Py co-infected group a significant decrease was observed only at 25 dpi. Py single infected animals also presented a significant drop in the percentages of CD8+T cells. In co-infected groups, the percentages of CD8+T cells were bellow controls at 10, 17, and 25 dpi. Low percentages of DP splenocytes were observed in Py single infected and co-infected groups, which were different form controls only when animals started to recover from malarial infection (17 and 25 dpi). On the other hand, P. yoelii infection increased the percentage of DN splenocytes during the entire experiment, and Leishmania co-infection was able to delay this enhancement. The percentages of CD4, CD8, DP, and DN splenocytes in Leishmania single infected groups (both Lb and La) remained similar to uninfected controls at all time points.

### DISCUSSION

Malaria and CL are neglected diseases spread over many tropical and subtropical regions of the World showing extensive overlapping geographic distribution. In addition, the increase in travel, migration and war refugees from endemic regions are factors that also contribute to the risk of simultaneous infections by Plasmodium spp. and Leishmania spp. parasites. Some cases of human Malaria and VL co-infection were described in the past and recently in different parts of the world (Yoeli, 1948; Ab Rahman and Abdullah, 2011; van den Bogaart et al., 2012, 2014; Bin Mohanna, 2015), demonstrating that coinfection may occur more frequently than we would expect. An increasing amount of evidence shows that co-infections may affect the natural outcome and progression of diseases due to the modulation of immune response (La Flamme et al., 2002; Kolbaum et al., 2012; Qi et al., 2013; van den Bogaart et al., 2014). In the present study, we established a co-infection model of American CL and a non-lethal murine Malaria and demonstrated that co-infection is able to affect the pathogenesis and outcome of both diseases.

Pilot studies were carried out in our Laboratory in which mice were co-infected at different times and orders. It was observed that the outcome of malaria or Leishmania infections were the same in any time and order of infections tested. For operational reasons we selected day 3 for Py infection. In addition, our results regarding the outcome of both diseases are similar to those published by Coleman et al. (1988a,b) in P. yoelii and L. amazonensis coinfection experiments.

Rodent Malaria parasites provide great models to investigate pathogenesis and immune mechanisms during infection (Li et al., 2001; Zuzarte-Luis et al., 2014). The non-lethal strain 17XNL of P. yoelii generally causes a transient disease with moderate parasitaemia, weight loss, splenomegaly, hypothermia, and anemia (Coleman et al., 1988b; Kobayashi et al., 1996; Niikura et al., 2008; Kolbaum et al., 2012; Karadjian et al., 2014). In our study, BALB/c mice infected with blood-stage P. yoelii 17XNL develop a self-limiting infection with mild parasitaemia that resolve in 4 weeks. Prior infection with L. braziliensis reduced parasitaemia, while animals previously infected with L. amazonensis showed a tendency to have higher parasitaemia than Py single infected group. Mortality during acute Malaria increased with L. amazonensis co-infection, demonstrating that the existence of intrinsic differences between this two Leishmania species during co-infection, may affect animal survival.

The course of experimental CL is dependent on a combination of factors: Leishmania and host species, parasite strain, site, and size of the inoculum among other aspects (Pereira and Alves, 2008; Pereira et al., 2009; Ribeiro-Gomes et al., 2014). The murine model of subcutaneous infection with L. major generated a great amount of data concerning the immune response in CL. It was in this model that could be demonstrated the importance of a type 1 (proinflammatory) specific immune response for healing the infection in genetically resistant strains of mice (such as C57BL/6), as well as the dominance of a type 2 immune response (with a strong production of IL-4 and IL-10) in susceptible BALC/c mice. On the other hand, all strains of mice that have been tested so far using the subcutaneous route of infection are susceptible to L. amazonensis, and non-permissive hosts for L. braziliensis.

The intradermal rout of infection with low doses of parasites in the ears, first developed for L. major infection (Belkaid et al., 2000), proven also to be an important experimental model for L. braziliensis studies. After intradermal infection in the ears with L. braziliensis promastigotes BALB/c are able to develop ulcerated lesions that heal spontaneously, similar to the most common clinical manifestation of American Tegumentary Leishmaniasis caused by L. braziliensis: the localized cutaneous form (de Moura et al., 2005; Falcão et al., 2012; Carregaro et al., 2013). In the present work, mice infected intradermally with L. braziliensis

developed a localized lesion that increased in size, became ulcerated and self-resolved by week 16 post infection, as expected (data not shown). Co-infected mice exhibited similar pattern in lesions development, but those were smaller and presented less ulcerations. L. amazonensis infection, on the other hand induced progressive non-healing lesions, and at the end of the study (11 weeks after L. amazonensis infection) 100% of the animals presented ulcerated lesions. Interestingly, during acute Py infection (in the 1st 4 weeks post L. amazonensis infection), coinfected group presented a transitory delay on the development of lesions as well as smaller lesions when compared to La single-infected mice. As mentioned before, the differences in susceptibility to Leishmania species in murine models, as well as in humans, have multifactorial causes, one of them being the type of immune response generated after infection. For this reason, the differences observed in the disease outcome of L. braziliensis and L. amazonensis infection during Malaria co-infection must be evaluated in the context of the intense systemic immune response elicited by P. yoelii 17XNL in the 1st weeks of Leishmania infection.

It is well described that Malaria erythrocytic stage triggers a strong IFN-γ response in both rodent and human Malaria (Shear et al., 1989; Chen et al., 2010; Perez-Mazliah and Langhorne, 2014). The first wave of proinflammatory cytokines is sustained by the innate immune system, when parasitaemia is still low, and is characterized by the release of IFN-γ, TNF-α, IL-2, and IL-12. The inflammatory environment sustains CD4+ T cell polarization to a Th1 phenotype that provides more IFN-γ for macrophages activation and control of parasitaemia (Artavanis-Tsakonas and Riley, 2002; Artavanis-Tsakonas et al., 2003). In our experiments, we were able to detect an early and pronounced increase of IFN-γ levels in the serum of P. yoelii 17XNL single infected and co-infected groups, mainly in the beginning of the infection, as extensively described in literature (Shear et al., 1989; Kobayashi et al., 1996; Shan et al., 2012; Karadjian et al., 2014). While the enhanced levels of IFN-γ in the serum was only transitory (5 and 10 days after P. yoelii 17XNL) the levels of TNF and IL-6 also increased in animals infected with P. yoelii 17XNL, but remained elevated for a longer period of time (during patent parasitaemia) in the serum. Co-infected groups presented smaller lesions than Leishmania single infected groups and this effect occurred simultaneously with the increased cytokines levels in the serum, during Malaria erythrocytic phase. The strong proinflammatory environment caused by P. yoelii 17XNL infection could explain the delay on lesions development in co-infected groups, given the fact that IFN-γ and TNF play an essential role in controlling Leishmania replication inside macrophages (Ashok and Acha-Orbea, 2014). Obviously a clearly scenario can be achieve if we evaluate the Leishmania-specific immune response developed in Leishmania single infected and Leishmania /P. yoelii 17XNL co-infected groups. This matter is currently under investigation.

In CL as well as in Malaria, excessive inflammatory response can lead to pathology or even death. Therefore, the balance between pro and anti-inflammatory cytokines is important to control pathology and parasitaemia (Artavanis-Tsakonas et al., 2003; Antonelli et al., 2005; Oliveira et al., 2014). Overall we could observe a tendency for reduced levels of IFN-γ, TNF, IL-6, and IL-10 in the serum of co-infected groups, when compared to P. yoelii 17XNL single infected group, suggesting a modulation of P. yoelii 17XNL induced immune response by Leishmania co-infection. On the other hand, L. braziliensis and L. amazonensis co-infection exerts opposite effects on P. yoelii 17XNL infection (**Figures 2** and **3**). IL-10 is a key regulatory cytokine to protect mice against pathology during acute Malaria (Kobayashi et al., 1996; Freitas do Rosario and Langhorne, 2012). Interestingly the reduction in the serum levels of IL-10 reached statistically significance only in L. amazonensis co-infected group at days 5 and 10 post P. yoelii 17XNL infection. This significant reduction in serum IL-10 levels could be linked to the persistent parasitaemia and deaths observed in L. amazonensis co-infected group (**Figure 2**).

Similar to several other parasitic diseases, experimental Malaria infections can cause severe thymic alterations (Savino, 2006; De Meis et al., 2012). During infection the organ undergoes a strong atrophy with disruption of its architecture that may influence T cell maturation. It has been already demonstrated that mice infected with P. chabaudi or P. berghei, two other rodent Malaria species, exhibit an important thymic atrophy with cellular depletion and histological disorganization (Seixas and Ostler, 2005; Andrade et al., 2008; Francelin et al., 2011; Lima et al., 2012). Thymic atrophy caused by infectious diseases may occur in combination with one or more of the following events: impairment of thymocyte proliferation, increase in thymocyte death and increase in thymocyte migration to peripheral lymphoid tissue (De Meis et al., 2012). Increase in apoptosis and thymocyte migration has been described in a variety of diseases (Savino, 2006), including Malaria (Francelin et al., 2011).

Accordingly, in P. yoelii 17XNL infection we observed a profound thymic atrophy with a reduction in the percentage of double positive cells (CD4+CD8+) and an increase in the percentage of single positive (CD4+ and CD8+) and double negative cells (CD4−CD8−). We also observed a high degree of apoptosis and necrosis in the thymocytes of P. yoelii single infected and co-infected groups, with no difference between groups (data not shown). Besides the intense atrophy during acute Malaria, thymus was capable to recover its normal size and cellularity after P. yoelii elimination, in accordance with observations with P. chaubaudi non-lethal infection (Seixas and Ostler, 2005). Infections with both Leishmania species were not able to cause thymus involution in mice, probably due to the localized nature of the cutaneous lesions they cause, with no or little impact in the systemic immune environment. Remarkably, thymus recovery occurred faster in co-infected animals, what we believe is a reflection of the minor inflammatory environment observed in co-infected animals.

The reduction in double positive thymocytes has also been described as common feature in infectious diseases, and can be explained by a premature escape of immature cells to blood and peripheral lymphoid organs (De Meis et al., 2012). P. yoelii 17XNL single infected and co-infected groups showed decreased percentages of double positive CD4+CD8+ splenocytes when compared to Leishmania single infected animals and uninfected controls, suggesting that the reduced percentages of double

positive cells in the thymus of those groups were not due to increased migration to the peripheral lymphoid organs.

Among the mechanisms described to clear blood-stage Malaria parasites are mature isotypes antibodies and antibodyindependent T cell mechanisms (Marsh and Kinyanjui, 2006; Beeson et al., 2008), but all of them require the activation of CD4+ T cells. In our model, CD4+ T cells presented a transitory increase at 10dpi but presented a significant drop on their percentages after 17 dpi when compared to controls. In L. amazonensis co-infected group they equalized controls only at 25 dpi, while in L. braziliensis co-infection they were lower them controls and Leishmania single infected group. The nature of this cells (e.g., if they are regulatory the cells or effector T cells), as well as the cytokines they produce after stimulation with Leishmania antigens or P. yoelii infected erythrocytes are under investigation.

Interestingly the percentages of double negatives CD4 and CD8 splenocytes increased in P. yoelii 17XNL infected groups, and Leishmania co-infection were able to delay this enhancement. We are also performing multiparametric flow cytometry experiments to better understand the phenotype of this CD3+CD4−CD8−cells, since they can be NKT cells or γδT cells. Both cell types are components of the innate immune system and have been proposed to play significant roles in the clearance of blood-stage malarial parasites (Stevenson and Riley, 2004; Urban et al., 2005).

NKT cells are innate-like lymphocytes that account for approximately 5% of T lymphocytes in the spleen. They posses both T cell and NK cells surface markers since they can express CD4 or CD8 co receptors on their surface, or neither one of them (double-negative phenotype) as well as the NK1.1 surface molecule (Balato et al., 2009). Primary P. yoelii infection with non-lethal strain 265BY is able to induce an organ-specific and heterogeneous NKT cells response (Soulard et al., 2007). Hepatic NKT cells consisted mainly of CD1d-dependent CD4+ and double negative NKT cells, whereas splenic NKT cells presented a CD1d-independent TCRhigh CD4high phenotype during infection (Soulard et al., 2007). It was also demonstrated that CD49b+ CD3+ natural killer T (NKT) cells increased in the liver after a primary infection with P. yoelii non-lethal strain (17XNL), and that CD1d-restricted NKT cells, which secrete IFNγ, are critical to reduce liver-stage burden in a secondary infection (Miller et al., 2014). Lack of type 1 IFN-alpha receptor signaling compromise the enhancement of NKT cells in the liver, showing a link between type I IFN signaling, cell recruitment, and subsequent parasite elimination.

Gamma delta T cells are the first to be generated in the ontogeny. They are able to respond quickly through the production of cytokines and can be divided into two types: interferon-producing cells (CD27hi) and IL-17-producing cells (CD27lo) (Zarin et al., 2015). In Malaria they are activated during pre-erythrocytic (liver) and erythrocyte stages and can produce INF-γ in vitro after stimulation with P. falciparum infected erythrocytes (Elloso et al., 1994; Farouk et al., 2004; Scholzen and Sauerwein, 2016). In BALB/c mice, the number of γδT cells increases considerably in the spleen during infection with P. yoelii non-lethal strain (17XNL) (Kopacz and Kumar, 1999), while this increase did not occur to the same extent in mice infected with the lethal strain of P. yoelii (17XL) suggesting that γδT cells play a protective role in Malaria (Kopacz and Kumar, 1999). In another work with P. yoelii non-lethal strain was observed an increase in the percentage of γδT cells in the spleen, liver and peripheral blood, peaking at day 21 of infection (Li et al., 2012). When the mice were depleted from γδT cells with monoclonal antibodies parasite elimination was delayed (Li et al., 2012).

Our results demonstrating an increase in CD3+CD4−CD8− cells during erythrocyte stage in P. yoelii 17XNL non-lethal strain infected animals both in the thymus (**Figure 7**) and in the spleen (**Figure 8**) does not discard the possibility that those can be either NK T or γδT cells, and the delay of this enhancement to occur in Leishmania co-infected groups when compared to P. yoelii single infected animals could have a correlation with the differences in cytokines production observed between P. yoelii 17XNL single infected and co-infected groups.

Taken together, our results suggest that coexisting infection with P. yoelii 17XNL and L. braziliensis or L. amazonensis may change diseases outcomes, and that Malaria outcome can be altered according to the Leishmania co-infection specie evaluated. On the other hand Malaria infection produced a transient delay on leishmanial lesions development. These alterations on Malaria and CL progress seem to be closely related to changes in the immune response as verified by alteration in serum cytokine levels and thymus dynamics during infection.

In areas where Malaria and CL are co-endemic, human populations are continuously exposed to Malaria and Leishmaniasis vectors bites and consequently are at risk of suffering repeated infections. These recurrent infections in endemic areas might cause permanent immunomodulatory effects, which can probably interfere in the outcome of both diseases. Moreover, the increase in Malaria parasitaemia, patency and mortality, observed in L. amazonensis/P. yoelii coinfection, might contribute to the severity of disease as well as to the maintenance of the malaria transmission in co-endemic areas. Considering the importance and the geographical distribution of Malaria and CL in tropical regions of the world, further investigation on immunomodulatory mechanisms during coinfection are necessary to understand how co-infection can affect not only the natural history and progression of diseases but also the treatment and prevention of both.

### AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: DB, PL, JF, and RP. Performed the experiments: RP, PL, DS, DV, DP, and DB. Analyzed the data: RP, PL, DV, DS, and DB. Contributed reagents/materials/analysis tools: DB, PL, and DV. Wrote the paper: RP, PL, and DB.

### FUNDING

This work was supported by the IOC/FIOCRUZ and PAEFFIOTEC (IOC-008-FIO-15-47 and IOC-008-FIO-15-66). The publication of this article was funded by IOC/FIOCRUZ. RP, DS, and DP are recipient of CAPES Doctoral Fellowships. DV and JF are recipient of CNPq Productivity Fellowships.

#### ACKNOWLEDGMENTS

fmicb-07-00982 June 24, 2016 Time: 16:57 # 14

We'd like to express our gratefulness to Professor Dr. Fabio T. M. Costa from Departamento de Genética, Evolução e Bioagentes

#### REFERENCES

from Universidade Estadual de Campinas-UNICAMP, SP/Brazil for providing us the P. yoelii 17XNL non-lethal strain utilized in this work. The authors also thank the Center for Laboratory Animal Breeding of the Oswaldo Cruz Foundation (CECAL) and the Center of Animal Experimentation of Leônidas Deane building for providing and keeping animals used in this study. We also thank the Flow Cytometry Core from IOC/FIOCRUZ.

Development of lesions in a Leishmania-susceptible (BALB/c) strain of mouse. Exp. Parasitol. 65, 269–276. doi: 10.1016/0014-4894(88)90131-2

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Pinna, Silva-dos-Santos, Perce-da-Silva, Oliveira-Ferreira, Villa-Verde, De Luca and Banic. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Galia Ramírez-Toloza1 \*, Paula Abello2 and Arturo Ferreira2 \**

*1 Faculty of Veterinary Medicine and Livestock Sciences, University of Chile, Santiago, Chile, 2Program of Immunology, Faculty of Medicine, Institute of Biomedical Sciences (ICBM), University of Chile, Santiago, Chile*

Eight to 10 million people in 21 endemic countries are infected with *Trypanosoma cruzi*. However, only 30% of those infected develop symptoms of Chagas' disease, a chronic, neglected tropical disease worldwide. Similar to other pathogens, *T. cruzi* has evolved to resist the host immune response. Studies, performed 80 years ago in the Soviet Union, proposed that *T. cruzi* infects tumor cells with similar capacity to that displayed for target tissues such as cardiac, aortic, or digestive. An antagonistic relationship between *T. cruzi* infection and cancer development was also proposed, but the molecular mechanisms involved have remained largely unknown. Probably, a variety of *T. cruzi* molecules is involved. This review focuses on how *T. cruzi* calreticulin (TcCRT), exteriorized from the endoplasmic reticulum, targets the first classical complement component C1 and negatively regulates the classical complement activation cascade, promoting parasite infectivity. We propose that this C1-dependent TcCRT-mediated virulence is critical to explain, at least an important part, of the parasite capacity to inhibit tumor development. We will discuss how TcCRT, by directly interacting with venous and arterial endothelial cells, inhibits angiogenesis and tumor growth. Thus, these TcCRT functions not only illustrate *T. cruzi* interactions with the host immune defensive strategies, but also illustrate a possible co-evolutionary adaptation to privilege a prolonged interaction with its host.

Keywords: calreticulin, *Trypanosoma cruzi*, trypomastigotes, complement system, C1q, cC1qR, tumor growth, immune response

#### INTRODUCTION

*Trypanosoma cruzi* (the protozoan agent of Chagas' disease) cell infection is preceded by a variety of molecular interactions (1). Of relevance is the generation of a synapsis involving parasite endoplasmic reticulum (ER)-resident *T. cruzi* calreticulin (TcCRT) that, after translocation, interacts with complement component C1. C1 is then inactivated and recognized by cC1qR (a membrane form of mammalian CRT). The complement system, an important arm of innate and adaptive immune responses, is thus inhibited and parasite infectivity increased.

A significant decrease in experimental tumor growth is observed in experimental animals treated with recombinant TcCRT (rTcCRT) or infected with *T. cruzi.* A unifying molecular basis for these apparently unrelated phenomena is proposed herein. These molecular interactions do provide benefits for both the host and the parasite.

Through evolution, microbial agents have developed different mechanisms to resist the host immune response. In apparently unrelated strategies, some infectious agents elicit antitumor

#### *Edited by:*

*Alexandre Morrot, Federal University of Rio de Janeiro, Brazil*

#### *Reviewed by:*

*Graciela Alicia Cremaschi, Institute of Biomedical Research (BIOMED), Argentina Celio Geraldo Freire De Lima, Federal University of Rio de Janeiro, Brazil*

#### *\*Correspondence:*

*Galia Ramírez-Toloza<EMAIL_ADDRESS>Arturo Ferreira<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 01 June 2016 Accepted: 27 June 2016 Published: 11 July 2016*

#### *Citation:*

*Ramírez-Toloza G, Abello P and Ferreira A (2016) Is the Antitumor Property of Trypanosoma cruzi Infection Mediated by Its Calreticulin? Front. Immunol. 7:268. doi: 10.3389/fimmu.2016.00268*

immune responses, leading to inhibition of cancer progression (2). Although these antitumor effects have been reported for several decades now, for a variety of infections, information on pathogen molecules involved is scarce (3).

Eight to 10 million people in 21 endemic countries are infected with *T. cruzi*. In about 30% of those infected, manifests, Chagas' disease, a worldwide neglected tropical chronic illness (4, 5). The disease, originally endemic in Latin America, is now global, mainly because of migrations to USA, Canada, Europe, Oceania, and Asia (6), where transmission is mainly through blood transfusions, organ transplants, or congenital (7).

Eighty years ago, it was proposed that *T. cruzi* possesses an anticancer activity. Several *T. cruzi* strains displayed growth inhibitory effects over multiple transplanted or spontaneous tumors, in animal experimental models and humans (8, 9). This property was attributed to a "toxic substance" secreted by the parasite (10, 11). This "toxin" reduced pain, tumor growth, bleeding, and local inflammation in humans affected by a variety of tumors (12).

Chronically infected rats are more resistant to a carcinoma induced by 1,2-dimethylhidrazyne (9), and *T. cruzi* has a tropism for tumor cells, suggesting an antagonistic relationship between Chagas' disease and cancer development (8). Elemental Darwinian reasoning allows us to propose that, if host survival is favored, chances for improved parasite persistence are evident.

Some authors have proposed that tumor and parasites compete for nutrients with consequent inhibition of tumor growth (13). However, this hypothesis is not entirely satisfactory since tumor growth is a multistep and complex process involving development of new blood vessels (angiogenesis) that provide the tumor with the necessary nutrients, oxygen, and means for waste removal (14). Other investigators have demonstrated, using a recombinant non-pathogenic *T. cruzi* clone as vector of a testis tumor antigen, the activation of T cell-mediated immunity. This specific cell immunity could delay tumor development in infected mice (15). In this work, it would have been important to define whether the non-pathogenic *T. cruzi* clone used translocatesexternalizes its CRT. Non-infective epimastigotes are strongly impaired in their capacity to translocate this chaperone (16). Moreover, hemiallelic *TcCRT* KO, wild type, and transgenic parasites, respectively carrying one, two, and three *TcCRT* gene copies, express increased levels of the protein, *in vitro* resistance to human complement, and higher infectivity (16, 17).

Most likely, multiple parasite molecules and mechanisms are involved in the tumor resistance mediated by *T. cruzi* infection. Understanding these mechanisms may contribute to identify new therapeutic targets against cancer and Chagas' disease.

Our laboratory has been working for more than 20 years now with TcCRT, a multifunctional ER-resident protein that the parasite translocates to the external milieu (as depicted in **Figures 1A,B**). TcCRT is involved in a multiplicity of

and kill tumor cells that also present TcCRT-derived peptides (I).

present these nTcCRT-specific peptides to cytotoxic T lymphocytes, thus leading to their activation. These CD8+ cytotoxic T lymphocytes will leave the lymph node

host–pathogen interactions. Thus, TcCRT is a potent virulence factor that inhibits the angiogenesis and a likely responsible, for at least in important part, of the antitumor effects of *T. cruzi* infection.

### IN EUKARYOTES, CALRETICULIN, AN ER-RESIDENT CHAPERONE PROTEIN, MEDIATES ANTITUMOR PROPERTIES

Calreticulin (CRT) is a 45 kDa protein, mainly residing in the ER (18). CRT participates in a variety of physiological and pathological processes in different cellular types (19). Thus, CRT contributes in multiple physiological processes such as control of glycoprotein folding quality system and binding to monoglucosylated high mannose glycans (20). Moreover, CRT is involved in quality control process during protein synthesis, including integrins, surface receptors, and transporters (21), and it is considered as an intracellular Ca2+ regulator (22).

Calreticulin is also found in the cytosol, nucleus, secretory granules, on the plasma membrane, and free in the extracellular milieu (18), accelerating cutaneous wound healing (23–25) and regulating cell adhesion by interacting with the cytosolic tail of the integrin alpha subunit (18); nuclear export of some steroid hormone receptors (26–28) and the stability or translation of a variety of RNAs (29–33). CRT is an mRNA binding protein that regulates mRNA stability (19).

Calreticulin also participates in the immune response against apoptotic cancer cells (34–38), and surface exposure of CRT participates as an "eat me" signal required for phagocytosis on dying tumor cells (39). Tumor tissues express significant higher levels of CRT compared to normal tissues (40). Indeed, its expression is related to the clinical stage and lymph node metastasis in several types of cancer (41, 42).

Over 40 functions have been described for human CRT (HuCRT) (43). These functions reside in three different domains: globular N-terminal (N), proline-rich (P), and acidic C-terminus (18). HuCRT and its N-terminal fragment bind laminin (44) with antiangiogenic properties *in vitro* and *in vivo* (45, 46) and inhibit the growth in several tumor models (47–49).

Vasostatin, a CRT 180 amino acid N-terminal fragment, is an endogenous inhibitor of angiogenesis and suppressor of tumor growth. It inhibits vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) proliferation and tube formation in Matrigel and induces cell apoptosis under oxygen deprivation (50).

Calreticulin is present in humans (51), insects (52, 53), nematodes (54–57), protozoans (58–61), and plants (62). A high identity is shared among CRTs from different species. Thus, *Onchocerca volvulus*, *Schistosoma mansoni*, and *Leishmania donovani* share 50% of the identity in amino acid sequence with HuCRT.

Examples of important evasive strategies performed by CRTs from different parasite species are *Amblyomma americanum* [secretes CRT during the feeding process (63)] and *Schistosoma cercariae* [uses CRT in the penetration of gland cells or skin and parasite migration (54)].

## HOW DOES *T. CRUZI* CALRETICULIN PARTICIPATE IN THE HOST–PARASITE INTERPLAY?

Given the important pleiotropic HuCRT behavior, the CRT model opens interesting research opportunities on how this protein, alone or interacting with others, intervenes in the host–parasite interactions.

For 25 years now, our laboratory has worked with TcCRT. This protein is coded by only one gene with a variable number of copies whose involvement in TcCRT expression will depend on the *T. cruzi* clone and strain studied (unpublished data). A TcCRT gene was cloned, sequenced, and expressed in our laboratory in 1991 (58). We identified variable low plasma levels of anti-native TcCRT antibodies in *T. cruzi*-infected humans (64), thus revealing the immunogenic capacity of the native protein.

*Trypanosoma cruzi* calreticulin also binds monoglucosylated glycans (60) and participates in the maturation of cruzipain, a lysosomal protease (65) present in *T. cruzi*. Although TcCRT locates mainly in the ER, it is also found in the Golgi complex, reservosomes, flagellar pocket, cell surface, cytosol, nucleus, and kinetoplast (66, 67). However, the mechanisms involved in these diverse TcCRT localizations are unknown. Thus, TcCRT, in spite of its KEDL-ER retention sequence [KDEL in mammal CRTs (18)], translocates from the ER to the extracellular environment (**Figures 1A,B**) where, besides inhibiting complement (66) and acting as a virulence factor (68), it mediates antitumor effects.

In spite of the long evolutionary distance, TcCRT still shares 50% of overall sequence homology with HuCRT, reaching up to 80% in critical functional domains. Moreover, the general globular N-domain, responsible of antiangiogenic properties and the structural features of the extended arm P-domain also share structure homologies, thus announcing the possibility of functional similarities (69).

Two important TcCRT functions may explain the relationship between *T. cruzi* infection and cancer. First, TcCRT is an important complement inhibitor (**Figure 1D**) and virulence factor (**Figure 1E**). Second, TcCRT inhibits angiogenesis (**Figure 1C**). Both functions are central to inhibit tumor growth.

## TcCRT IS AN IMPORTANT VIRULENCE FACTOR IN *T. CRUZI*

Similar to HuCRT (70, 71), TcCRT inhibits the complement system by interacting with C1 (**Figure 1D**), the first component of its classical pathway (66, 72–74). TcCRT is translocated from the ER to the area of flagellum emergence (**Figure 1A**) (66), where C1 is recruited by parasite-bound TcCRT and inhibited at the earliest complement activation step (C4b generation) (**Figure 1D**). TcCRT also affects the ability of C1s to activate C4, in a calcium-independent manner (74). Inhibition of C1 is a significant complement evasion strategy, with consequences in the host–parasite relationships. Although HuCRT and TcCRT prevent binding of the serine proteases to C1q, they do not displace the serine proteases from the preformed stabilized C1 (C1q, r2, and s2) complex (74). TcCRT also binds to MBL and Ficolins (75). C1, MBL, and Ficolins are three complement "danger signal" recognition macromolecular modules present in plasma. These molecular complexes are genetically, structurally, and functionally related, but they differ in the nature of the recognized danger signals (76). More recently, we have proposed that L-Ficolin binds TcCRT, inhibiting the lectin pathway. This inhibition may represent other *T. cruzi* strategy to inhibit the host immune response (75). In agreement with these findings, TcCRT is present on the parasite surface co-localizing with C1q (66).

Human CRT is also a membrane receptor for C1q [cC1qR (77)], and it may bridge TcCRT on the parasite surface with HuCRT present on the host cell (**Figure 1E**) (78). The TcCRT/ C1q/HuCRT synapsis represents the culmination of an important molecular mimicry strategy. Apoptotic cells to be phagocytized use a similar mechanism (34, 36, 37). The CRT/C1q complex is recognized as an "eat me" signal by cC1qR on phagocytes. This signal is also used by *T. cruzi* as an "apoptotic mimicry" strategy (i.e., by capturing C1 in the area of flagellum emergence), thus facilitating the invasion/infectivity of host cells (79). This TcCRT-C1q-mediated parasite infectivity correlates with significant increases in TcCRT mRNA levels during early (cell contact and penetration) infection stages (36, 66, 68, 69, 72, 79). The TcCRT– C1q interaction can be prevented with anti-TcCRT F(ab′)2 fragments (devoid of the C1-binding Fc domains) (80). Indeed, passive immunization of mice with these fragments decreases infectivity (68). Congenital transmission is an important *T. cruzi* transmission pathway. Human pregnancy is a condition of elevated circulating CRT (81, 82). Moreover, human placenta expresses high CRT levels (83). We have recently proposed that the TcCRT/C1q/HuCRT interaction is very important in an *ex vivo* model of infection of human placenta (84), indicating a possible mechanism to explain the congenital transmission.

### TcCRT PARTICIPATES IN THE INHIBITION OF TUMOR GROWTH

Cancer is omnipresent in human history, and it also affects most of the living animal species, as a natural phenomenon of sporadic cellular dysfunction. Mammary, prostate, lung, cervix/uterine are just a few examples of cancer that, taken together, have epidemic proportions.

Interestingly, in patients infected with *T. cruzi*, cancer is rare (10, 12). About 80 years ago, Roskin, Ekzempliarskaia, and Klyuyeva, researchers from the former Soviet Union, postulated an experimental anticancer toxic activity derived from this infection. When they inoculated *T. cruzi* extracts, directly in a peritumoral area, in different tumors, both in experimental animals and in humans, similar results related to reduction of tumor size were obtained (10–13, 85, 86). More recently, the parasite capacity to infect preferentially tumor cells, as compared to normal host cells, was described (8). Although, in general, these data suggest an antagonism between *T. cruzi* infection and tumor growth (8), and research progress in these areas was seriously hampered by the intense international political problems of those years (i.e., the Cold War) (11). Although several publications on these issues have appeared during the last decades, the molecular basis of this phenomenon has remained elusive.

We propose that TcCRT is an important mediator of the antitumor effects of *T*. *cruzi* infection. Similar to HuCRT, TcCRT is antiangiogenic in *in vitro*, *ex vivo*, and *in vivo* models (**Figure 1C**) (3, 87, 88). Moreover, TcCRT inhibits the growth of a mammary adenocarcinoma and a melanoma in different experimental animal models (3, 87–89). The inhibition of tumor angiogenesis was proposed as a cancer therapy almost 40 years ago (90). For this reason, molecules or drugs with capacity to inhibit angiogenesis are currently applicable to a wide variety of tumors, often as a complement to other therapies (91).

*Trypanosoma cruzi* calreticulin and its N-terminal domain (N-TcCRT) were studied in different experimental set ups in mammals, *Homo sapiens* included (3). Thus, rTcCRT and its N-TcCRT inhibit capillary growth *ex vivo* in *Rattus rattus* aortic rings, morphogenesis, proliferation, and chemotaxis in human umbilical cord endothelial cells (HUVECs) (3) and *in vivo* angiogenesis in the *Gallus gallus* chorioallantoid membrane (CAM) assay (87). TcCRT was overall more effective, in molar terms, than HuCRT (3). Interestingly, in the CAM assay, the antiangiogenic TcCRT effect was fully reverted by polyclonal antibodies against rTcCRT (88).

In agreement with the previously described facts, the *in vivo* antitumor capacity of *T. cruzi* infection is paralleled by the inoculation of rTcCRT, with inhibits by 60–70% the time-course development of a murine mammary methotrexate multiresistant adenocarcinoma (TA3-MTX-R) (7).

### *T. CRUZI* INFECTS NEOPLASTIC CELLS AND PROMOTES AN IMMUNE RESPONSE

Native TcCRT (nTcCRT) on the parasite contacts ECs, mediating internalization of *T. cruzi* and inhibition of tumor growth. This nTcCRT/EC contact may be indirect, mediated by C1q (**Figure 1E**) or by direct binding to scavenger receptors (SRs) (**Figure 1F**). TcCRT has affinity for collagenous structures, a possible explanation for its binding to human C1 and to SRs (66, 68). Fluid-phase Fucoidan, bearing extensive collagen-like sequences, inhibits the binding of CRT to SR-A present on both phagocytic cells (92) and the internalization of TcCRT by ECs (3).

### IS NATIVE TcCRT RESPONSIBLE FOR THE ANTITUMOR EFFECT OF *T. CRUZI* INFECTION?

Recombinant TcCRT has important *in vivo* antiangiogenic and antitumor activities (3, 88). The antitumor effect of *T. cruzi* extract has been recently reproduced in a rat model. Experimental animals showed a strong cytotoxic response against tumor, with activation of CD4+ and CD8+ T cells and splenocytes. Moreover, a humoral adaptive immune response is generated. These anti-*T. cruzi* antibodies cross-reacted with tumor cells, inducing an antibody-dependent cellular toxicity *in vitro* (93). In a mouse model, we have reverted the antitumor effect of a *T. cruzi* epimastigote extract with specific antibodies against rTcCRT. Moreover, anti-rTcCRT F(ab′)2 antibodies (devoid of their capacity to interact with C1) neutralize the antitumor activity of *T. cruzi* infection, thus identifying nTcCRT as a mediator of this effect (unpublished data).

### HOW DOES TcCRT INHIBIT TUMOR GROWTH IN INDIVIDUALS INFECTED WITH *T. CRUZI*?

We propose that, during *T. cruzi* infection, nTcCRT mediates key alterations in the tumor cell microenvironment leading to an adaptive immune response, with significant antitumor effects. Once in the circulation, *T. cruzi* must swiftly invade ECs (**Figures 1E,F**). Translocated-exteriorized TcCRT (**Figures 1A,B**) (92) will recruit and inactivate plasma complement C1 (**Figure 1D**) and inhibits angiogenesis (**Figure 1C**). This will allow the parasite to contact ECs *via* cC1qR (**Figure 1E**) (77, 94). Otherwise, the chaperone protein could interact directly with SR-A1 on ECs (**Figure 1F**) (95–97). Both pathways may lead to antiangiogenesis and generate a stressful environment where tumor cells will externalize their CRT, as previously shown with other stressing agents, such as Antracyclins (37). C1 recruitment and increased tumor cell phagocytosis by dendritic cells will follow (**Figure 1H**).

On the other hand, an adaptive immune response may be invoked by inoculated TcCRT or by its native counterpart timely externalized by infecting trypomastigotes (66) or present in epimastigote extracts (75). The chaperone protein should reach the surface of tumor cells (or ECs), thus generating a site for C1 binding (**Figure 1G**), followed by phagocytosis of these complexes by dendritic cells (**Figure 1H**). Targeting these activities on tumor cells should be favored by the parasite tropism for these tissues. The relevant novelty of parasite TcCRT is its difference in amino acidic sequence with the mammal (murine, in this case) counterpart. This difference may reach 50%, while mammal CRTs differ among them by no more than 10% (73). Upon arrival to the regional lymph nodes, these dendritic cells will present antigenic peptides derived from TcCRT, thus activating cytotoxic T lymphocytes, among other possibilities. Whether tumor cells can cross-present peptides derived from endocytosed TcCRT to cytotoxic T cells (**Figure 1I**) is a matter of current research in our laboratory. Activated cytotoxic T cells should then return to the tumor site and act against neoplastic tumor cells. Activation of CD4+ T cells *via* MHC II presentation, with stimulation of B cells and resulting ADCC against tumor cells, is a possibility that should also be entertained.

### REFERENCES

In our murine models, these antitumor effects are better performed by TcCRT, as compared to HuCRT. Among mammals, CRTs are at least 95% homologous in amino acidic differences. CRT immunogenicity across mammal species is thus restricted. On the other hand, because of extensive evolutionary distances, TcCRT amino acidic sequence differs by 50% with its mammal counterparts. Thus, TcCRT is more capable of generating immunogenic epitopes on the surface of mammal tumors. Recently, the expression of CRT has been correlated with a favorable prognosis of cancer. The high expression of CRT on tumor cells has been associated with a high density of infiltrating mature dendritic cells and effector memory T-cell subsets, suggesting that CRT triggers the activation of an adaptive immune response in the tumor microenvironment (98). Thus, TcCRT expressed and secreted by the parasite may be also important in this regard.

### CONCLUDING REMARKS

Infection with *T. cruzi* correlates with increased resistant to tumors. Since, during infection, nTcCRT is translocated to the parasite exterior and experimental parenteral administration of rTcCRT mimics the antitumor effects of the infection, nTcCRT is the most likely responsible molecule for these effects. Moreover, the antitumor effects of parasite infection can be specifically reverted by anti-rTcCRT antibodies. Since, in a large set of experimental animals treated with rTcCRT, no clinical deleterious effects have been detected by standard clinical veterinary criteria, we can now propose that rTcCRT or derived domains are interesting immunological tools to be considered in more advanced preclinical trials (e.g., rTcCRT capacity to bind to human mammary tumor cell lines *in vitro*, to subsequently incorporate C1, with increased capacity to induce phagocytosis).

### AUTHOR CONTRIBUTIONS

GR-T, PA, and AF designed experiments. GR-T and PA performed experiments. GR-T, PA, and AF interpreted the data. GR-T, PA, and AF generated key reagents. GR-T, PA, and AF wrote, revised, and edited the manuscript. GR-T, PA, and AF approved the manuscript.

### FUNDING

This work was supported by CONICYT-CHILE grants: FONDECYT Regular 1130099 and FONDECYT-Iniciación 11110251.

accumulation of antitumor immune cells and favorable prognosis. *Cancer Res* (2016) 76:1746–56. doi: 10.1158/0008-5472.CAN-15-1142

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Ramírez-Toloza, Abello and Ferreira. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Dendritic Cells: A Double-Edged Sword in Immune Responses during Chagas Disease

Natalia Gil-Jaramillo1 , Flávia N. Motta1,2, Cecília B. F. Favali3 , Izabela M. D. Bastos1 and Jaime M. Santana1 \*

1 Laboratório de Interação Patógeno-Hospedeiro, Instituto de Biologia, Universidade de Brasília, Brasília, Brazil, 2 Faculdade de Ceilândia, Universidade de Brasília, Brasília, Brazil, 3 Laboratório de Biologia do Gene, Instituto de Biologia, Universidade de Brasília, Brasília, Brazil

Dendritic cells (DCs) are the most important member of the antigen presenting cells group due to their ability to recognize antigen at the infection site and their high specialized antigen internalization capacity. These cells have central role in connecting the innate and adaptive immune responses against Trypanosoma cruzi, the causative agent of Chagas disease. These first line defense cells modulate host immune response depending on type, maturation level, cytokine milieu and DC receptor involved in the interactions with T. cruzi, influencing the development of the disease clinic forms. Here, we present a review of DCs–T. cruzi interactions both in human and murine models, pointing out the parasite ability to manipulate DCs activity for the purpose of evading innate immune response and assuring its own survival and persistence.

#### Edited by:

Alexandre Morrot, Federal University of Rio de Janeiro, Brazil

#### Reviewed by:

Uwe Ritter, University of Regensburg, Germany Marisa Mariel Fernandez, University of Buenos Aires, Argentina

> \*Correspondence: Jaime M. Santana<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology

Received: 01 June 2016 Accepted: 27 June 2016 Published: 14 July 2016

#### Citation:

Gil-Jaramillo N, Motta FN, Favali CBF, Bastos IMD and Santana JM (2016) Dendritic Cells: A Double-Edged Sword in Immune Responses during Chagas Disease. Front. Microbiol. 7:1076. doi: 10.3389/fmicb.2016.01076 Keywords: chagas disease, Trypanosoma cruzi, dendritic cell, immunoregulation, evasion strategy

## INTRODUCTION

More than a century has passed since Carlos Chagas discovered the pathogen Trypanosoma cruzi and the natural way of its transmission to humans and animals, and elucidated the corresponding human disease it causes (Kropf and Sá, 2009). Chagas disease is also transmitted through blood transfusions or organ transplants, vertically from mother to child through the placenta, and through contaminated food (Andrade et al., 2014). This neglected disease affects predominantly deprived people and induces social and economic impacts by decreasing patient's productivity and earning capacity (Hotez et al., 2007).

With 28,000 new cases per year and 8,000 newborns infected during gestation, Chagas disease affects about 8 million people, beyond 65 million live in areas of exposure and are at risk of contracting the disease (World Health Organization, 2015). Despite the efforts to reduce Chagas disease and the number of infections over the last two decades, it is still endemic in Latin America (Rassi et al., 2010; Strasen et al., 2013) and, as result of global migration, the number of people infected with T. cruzi is increasing in North America, Europe, Japan and Oceania (nonendemic areas) (Schmunis, 2007). Since Chagas disease crosses boundaries and spreads, it becomes not only a burden for the endemic countries but a worldwide health concern (Andrade et al., 2014).

Treatment of Chagas disease is one of the greatest therapeutic challenges in tropical medicine since the only drugs approved for human treatment − Nifurtimox and Benznidazole − date from the early 1970s and have carcinogenic properties conferred to nitrofuran and nitroimidazole, their active chemical groups, respectively (Bern, 2011; Wilkinson et al., 2011). Nevertheless, both

medicines share similar features: necessity of prolonged administration, effectiveness related to the acute phase and different susceptibility among T. cruzi strains (Bermudez et al., 2015). Aditionally, Nifurtimox prescription has been abolished in Brazil, Argentina, Uruguay, Chile and US due to its toxic effects over central nervous system, genotoxicity, and reduced efficacy (Wilkinson et al., 2011).

The natural disease progression consists of an acute phase that can be followed by an asymptomatic indeterminate phase, which represents most of the cases and, one-third of infected population progress to the chronic phase that may lead to death (Machado et al., 2013). Early clinical manifestations include headache, fever, and cough, which are non-specific signs; consequently most of infected individuals are neither notified nor treated. Symptomatic chronic stage of the disease usually occurs from 10 to 25 years after infection and typical manifestations are mild to severe cardiomyopathy and/or dilated digestive tract (megaoesophagus and megacolon) (Steverding, 2014). The mechanisms responsible for patient progression from the indeterminate to the symptomatic chronic phase are not completely understood, although the immunological events initiated during the acute phase undoubtedly drive the organism toward the development of a protective or deleterious immune response (Andrade et al., 2014).

It is unanimity that during the acute phase, dendritic cells (DC), along with macrophages, and natural killer (NK) cells, guarantee the host first line of defense against the parasite (Watanabe Costa et al., 2016). Guide by this context, our purpose is to present a review about DC–T. cruzi interaction pointing out the parasite ability to evade innate immune response to assure its own survival and persistence.

### Trypanosoma cruzi

Trypanosoma cruzi is an obligate intracellular protozoan of the Kinetoplastea Class, characterized by the presence of one flagellum and a single mitochondrion comprising the kinetoplast, a specialized DNA-containing structure. The Order Trypanosomatida comprises other parasites responsible for severe diseases in humans and other species, for instance Leishmania sp. (leishmaniasis), Trypanosoma brucei (African trypanosomiasis), Phytomonas sp. (plant diseases), and Crithidia sp. (arthropods diseases) (Stevens, 2008), that show adaptability toward their hosts with numerous sophisticated immune evasion strategies (D'Avila-Levy et al., 2015).

Trypanosoma cruzi strains present diversity in morphology, infection capacity, cell surface predominant antigens and other biological and biochemical features (de Lana et al., 2010). In 2009, the Second Satellite Meeting held in Buzios, Brazil suggested six Discrete Typing Units (DTUs) for classifying the several T. cruzi strains, named TcI to TcVI (Zingales et al., 2009). A DTU is a population set that is more genetically related among themselves than to any other population, showing common genetic, immunological and molecular markers (Tibayrenc, 1998). These DTUs subsets have different distribution among the American continent due to parasite adaptations to different vectors and reservoirs (Zingales et al., 2012), and, although, some characteristics are shared within the same DTU, differences in many aspects can be found among strains. T. cruzi Colombian and G strains, belonging to TcI group, are a clear example of this intra-DTU diversity. Colombian strain, isolated from humans, is highly infective (Ramírez et al., 2010), but no human infections were reported from G strain, which was isolated from anal gland secretions of an opossum (Deane et al., 1984). T. cruzi G strain has gp35/50 as predominant surface glycoprotein that appears to be related to its poor internalization by humans cells (Yoshida, 2006).

T. cruzi life cycle alternates between reduviid bug vectors and mammal hosts. Domestic and wild animals like opossum, bats, armadillo, and monkey may also act as reservoir host (Coura et al., 2002). The parasite presents different forms during its life cycle – epimastigotes remains in the insect gut; non-divinding and infective metacyclic trypomastigotes (MT) find in the insect feces and/or urine; bloodstream trypomastigotes that can circulate in the mammalian blood and, finally, the intracellular, proliferative and rounded amastigote form. A triatomine insect picks up the parasite trypomastigote forms by feeding on the blood of an infected mammal (Teixeira et al., 2009) and, once inside the vector, those forms differentiate into epimastigotes and multiply in midgut (Manchola et al., 2015). After migration to the bug's hindgut, epimastigotes attach to the waxy gut cuticle by their flagella and differentiate into infectious MT, which will be deposited along with feces/urine on the skin of the victim (Muñoz-Saravia et al., 2012). The parasite penetrates the new host through lesions caused by its bite or a variety of mucosal membranes. MTs invade host cells entering a parasitophorous vacuole (Romano et al., 2012), which fuses to lysosomes. Once free in the cytoplasm, the parasite differentiates into amastigotes that undergo binary fission multiplication and transform back to trypomastigotes, which are released upon rupture of the host cell membrane and infect neighboring cells or enter the bloodstream (Stecconi-Silva et al., 2003; Tonelli et al., 2004). Bloodstream trypomastigotes may begin another infection cycle when they are taken in the blood feeding of the vector (Bern, 2015). Humans and animals can be infected orally through the ingestion of food and drink contaminated by crushed infected insect vectors or their feces (Nóbrega et al., 2009; Bastos et al., 2010).

Immune evasion strategies developed by T. cruzi are essential to the establishment of a long-life infection. Complement inactivation, escape from phagolysosome, antibodies with no T-cruzi specificity and delayed immune response are some examples of those strategies (Reviewed by Nardy et al., 2015; Cardoso et al., 2016). Another mechanism involved in parasite persistence is the manipulation of DC functions, which impairs an adequate host immune defense.

### DENDRITIC CELLS

Dendritic cells are bone-marrow-derived cells that belong to the antigen presenting cells (APC) group, being considered the most important member due to their high capacity to recognize and

internalize antigens at the infection site (Haniffa et al., 2009; Collin et al., 2013). They link innate and adaptive immune responses by capturing, processing and expressing antigens in the cell surface membrane (Planelles et al., 2003; Steinman, 2007). Immature DCs have a wide range of innate receptors that enables the recognition of pathogens via pattern recognition receptors (PRRs), which activate DCs through signaling pathways eliciting their maturation (Pearce and Everts, 2015). Toll-like receptors (TLRs), abundant in APC, are one of the bestcharacterized PPRs and efficiently detect pathogen-associated molecular patterns (PAMPs) that are located on the cell surface or in the lumen of endosomes. The presence of TLRs on the cell surface or in the lumen of endosomes enables efficient pathogen recognition and the development of an adequate innate immune response; i.e., TLR2, located in DCs plasma membrane, senses various components of pathogens and its stimulation induces the production of various proinflammatory cytokines. TLR9, located in DCs endolysosome, is involved in virus, bacteria, protozoa nucleic acid recognition and its activation also leads to the production of proinflammatory cytokines (Takeuchi and Akira, 2010). After antigen recognition, DCs can travel along the body from peripheral tissues to lymphoid organs/tissues where they present the processed antigens through their major histocompatibility complexes (MHC) to T cells (Lipscomb and Masten, 2002). Maturation process comprises differentiation from antigen-capturing specialized cells to presenting and stimulating specialized cells. Mature DCs can be identified by morphological aspects like cytoplasmic extensions and abundant intracellular structures (lysosomes, endosomes, granules, etc.) related to antigen processing and by modulation of molecular markers such as up-regulation of CD83, of co-stimulatory molecules like CD80 (or B7-1) and CD86 (or B7-2) and MHC (O'Neill et al., 2004).

Dendritic cells present antigens to lymphocytes CD8+ and CD4+ T by MHC class I and MHC class II, respectively (Blum et al., 2013). For MHC class I molecules, these antigens originate from intracellular sources; for MHC class II, from exogenous sources. Some DCs have an atypical ability, called cross-presentation that allows to load peptides from exogenous antigens onto MHC class I molecules (Vyas et al., 2008; Neefjes et al., 2011; Segura and Amigorena, 2015). MHC class I molecules are expressed by all nucleated cells and their antigen presentation pathway consists in a series of reactions: (1) intracellular proteins are degraded by the proteasome; (2) the peptides are delivered to the endoplasmic reticulum by the transporter associated with antigen processing complex; (3) antigens are loaded onto MHC class I molecules; (4) peptide–MHC class I complexes are transported via the Golgi to cell surface for presentation to CD8+ T cells (Vyas et al., 2008; Neefjes et al., 2011). Recently it was demonstrated that infection of HeLa cells with T. cruzi Y strain promotes a down-regulation of the immunoproteasome subunits biosynthesis as well as the MHC class I molecule expression, which could be considered a mechanism of parasite persistence inside the cell (Camargo et al., 2014). Unlike MHC class I expression, MHC class II are mainly expressed by APCs such as DCs, macrophages and B cells (Vyas et al., 2008; Neefjes et al., 2011). Extracellular antigens are taken up by APCs and placed into the phagosome. This compartment fuses with lysosomes to form phagolysosomes, where MHC class II molecules interact with the antigens. Peptide-loaded MHC class II molecules are then transported to the cell surface where they engage antigen-specific CD4+ T cells (Vyas et al., 2008; Neefjes et al., 2011). Curiously, MHC II molecules are in constant recycle and degradation process in immature DCs, but mature DCs exhibits a stable and prolonged antigen presentation (den Haan et al., 2014). It is worth mentioning that DCs antigen presentation is not enough for T lymphocytes activation and proliferation. Co-stimulatory molecules expression and cytokine production are also required and they are efficiently provided by mature DCs (den Haan et al., 2014). Following activation via TLRs, DCs may produce acute innate cytokines involved in local and systemic responses such as IL-1β, IL-6, IL-8, IL-12, and TNF-α (Verhasselt et al., 1997; Holdsworth and Can, 2015), however, DCs, under specific conditions, are also able to produce IL-10 and TGF-β for directing a regulatory response (**Table 1**).

Different DC subsets and maturation levels can produce distinct kinds of cytokines or co-stimulatory molecules that can lead either to an inflammatory or a regulatory response. Among DC subsets, myeloid (mDC or classical DC), plasmocytoid (pDC), Langerhans cells (LCs), and derived from monocytes (monocyte DC) are some well-known examples. In murines, mDCs are composed of two main groups: resident and migratory, which are further divided into two subsets: Batf3-dependent and IRF4-dependent DCs (Reviewed by Segura, 2016). mDC Batf3 dependent/migratory expresses CD11c, Clec9A, XCR1, CD103, and CD207; the resident one expresses CD11c, Clec9A, XCR1, and CD8. On the other hand, mDC IRF4-dependent/resident expresses CD11c, CD11b, CD172a; the same markers are found in the migratory subsets along with CD206. The major markers for pDC are CD11c, Ly6c, B220 and SiglecH. For monocyte DC, they are CD11c, CD11b, CD64, FcεRI, CD206, CD14, CD172a, and Ly6c (Reviewed by Segura, 2016). Finally, LCs is a special DC population present in epidermis and other stratified squamous epithelia, such as oral and genital mucous membranes and bronchus. Despite of being associated with these tissues, LCs may differentiate into migratory cells for antigen presentation; their principal markers are CD11c, CD207, EpCAM, and E-cadherin. Human mDCs classical markers are CD1c+, Dectin 1 (CLEC 7A), and Dectin 2 (CLEC6A). Regarding pDCs, CD303 (CLEC4C), CD304 (neuropilin), and CD123 (IL-3R) are human usual markers. Monocyte DCs may express CD14, CD209 (DC-SIGN), CD16, and CD1c (Reviewed by Collin et al., 2013). Lewis and Reizis (2012) have proposed the existence of two more kinds of DCs within these subsets: "receptors", cells more specialized in capturing antigens and producing cytokines and "presenters", cells that benefit from these cytokines and travel to lymph nodes for presenting the antigens. It represents another level of specialization among DCs, stating their diversity and their capacity to guide polarity, magnitude and specificity of immune responses (Lewis and Reizis, 2012).

Some studies have demonstrated that acute and chronic phases of Chagas disease require different polarizations of

immune response: Th1 profile is protective in an acute state and regulatory responses are important in preventing the chronic phase (Andrade et al., 2014; Dutra et al., 2015). For this reason, DCs are a key group of cells in Chagas disease: they could modulate response depending on type, maturation level, cytokine milieu and DC receptor involved, having a fundamental role in the development of the disease clinic forms including the undetermined stage.

### DENDRITIC CELL−T. cruzi INTERACTION: MURINE MODELS

Studies on acute Chagas disease in humans are limited due to lack of unique symptoms that characterize this disease state. Nonetheless, extending our knowledge about the acute phase is important because immunological events that take place in this stage have a great influence on the possible development of the chronic phase (Andrade et al., 2014). In this context, experimental murine infection may mimic the human disease, giving us a similar view on what happens at the beginning of T. cruzi infection.

Unlikely the habitual DCs response during an infection, the expression of important surface molecules like MHC, CD80 or CD86 can be reduced when DCs recognize T. cruzi antigens in a strain dependent manner, limiting DC maturation and antigen presenting capacity. During acute phase, splenic DCs infected by T. cruzi Tehuantepec strain shows low expression of CD86 molecules and are not able to migrate toward lymphoid organs/tissues (Chaussabel et al., 2003). T. cruzi high virulent RA strain (TcVI) induces bone-marrow DCs downregulation of cytokine production and of endocytic capacity added to a suppression of MHC class II, compared to non-infected cells (Poncini et al., 2008). These data are in concordance with the regulation studied by Alba Soto and coworkers, where they also detected a diminished MHC II expression in infected splenic DCs by the same T. cruzi strain. Additionally, they showed that the DC manipulated behavior induced by RA strain is not repeated for non-virulent K98 T. cruzi strain (TcI) (**Figure 1**; Alba Soto et al., 2003). Another comparative infection study, using mDCs, was performed using TcI (AQ1.7 and MUTUM) and TcII strains (1849 and 2369). The results demonstrated that both T. cruzi DTUs may modulate DCs to different extents and this modulation varied more between strains than between DTUs themselves. In general, both DTUs induced the production and expression of anti-inflammatory molecules, such as IL-10, production and PD-L1 and TLR2 expression. Regarding TLR2 expression, it seems that T. cruzi has molecules that bind this receptor promoting the production of anti-inflammatory cytokines such as IL-10. Oppositely, proinflammatory molecules did not present a pattern, varying depending on the strain. Finally, they also demonstrated that DC expression of differentiation and activation molecules was not polarized, which suggests that each strain of T. cruzi has possibly evolved specific evasion strategies (da Costa et al., 2014). Interestingly, when two mice lineages were infected by high virulent T. cruzi Y strain (TcII), the susceptible one showed splenic DCs with reduced capacity of antigen presentation and lower expression of CD40 and CD86 molecules compared with resistant lineages (Planelles et al., 2003). It is well known that the deficiency of co-stimulatory signals during crosspresentation may reduce T cell stimulation or lead to an anergic state (Boussiotis et al., 1996). Thus, it seems that impaired function by DC maybe helps parasite to evade the host immune system.

Cytokine production is another important aspect during DCs−T. cruzi interaction, particularly the production of IL-12, considered a protective molecule during the acute infection once it drives a polarized Th1 adaptive response that enables the host to adequately control parasite growth through IFN-γ (Andrade et al., 2014). In concordance, DCs from T. cruzi-resistant mouse lineages overexpressed IL-12 and TNF-α while the susceptible lineages produced the Th2 cytokine IL-4 (Planelles et al., 2003). IL-4 is known to mediate host susceptibility to T. cruzi but is also required for preventing immune hyperactivity and organ immunopathology (Abrahamsohn and Coffman, 1996; Laucella et al., 1996). Terrazas et al. (2011) demonstrated the protective role of IL-12 through T. cruzi Queretaro (TcI) strain infection of MIF (macrophage migration inhibitory factor)-deficient mice. MIF is a pleiotropic cytokine produced by multiple different cell types, including DCs that modulates the expression of several proinflammatory molecules (Cooke et al., 2009). It seems that it favors DC maturation through IL-12 secretion and activation of p38 MAPK protein, a kinase already known to be involved in DC maturation. On the other hand, MIF-deficient mice showed lower levels of IL-12 production and immature bone-marrow DCs, leading to a susceptibility of T. cruzi Queretaro (TcI) strain infection (Terrazas et al., 2011). The regulatory IL-10 cytokine is also associated with host susceptibility during acute stage of Chagas disease by limiting DCs induction of antimicrobial effector mechanism such as suppressing DC trafficking to draining lymph nodes (Corinti et al., 2001; Demangel et al., 2002; Alba Soto et al., 2010). A single intravenous injection of IL-10 deficient DCs that were pulsed with parasite antigens conferred an effective control against a lethal challenge with T. cruzi RA strain in mice. IL-10 deficient DCs were high Th1 cytokines producers and inducers of antigen-specific T lymphocytes after immunization (Alba Soto et al., 2010). On the other hand, Poncini et al. (2010) have showed that interaction of DCs with T. cruzi trypomastigotes was not able to activate the DCs, and these cells became TGF-β and IL-10 producers and were not efficient as lymphocyte stimulators, being classified as regulatory DCs. Furthermore, mature DCs have the capacity to induce NK cells activation and proliferation. NK cells play a significant role in innate immune response and surveillance as a result of their cytokine production and cytolysis of infected cells. Moreover, NK cells secrete IFN-γ, TNF-α, and GM-CSF, which promote DC maturation and activation of T-cell (Gerosa et al., 2002; Piccioli et al., 2002). Batalla et al. (2013) used RA and K98 T. cruzi strains to demonstrate the role of NK cells in regulating the maturation level of DCs. During both infections, NK cell was functionally activated and produced IFN-γ but also IL-10; NK cells from mice infected with T. cruzi RA strain (high virulence) exhibit reduced ability to lyse and fail to induce maturation

of bone marrow-derived immature DCs. This unbalanced DC population could difficult T cell stimulation for an adequate response. Finally, that IL-10 production observed by NK cells after infection with T. cruzi RA strain might lead to parasite persistence but can also limit the induction of a vigorous tissue-damaging T-cell response (Batalla et al., 2013). Cytokine production may also be modulated by parasite initial inocula. Three different inocula of T. cruzi Colombian strain were used to infect mice, resulting in differential expression of IFN-γ, IL-17, TNF-α, IL-4, and IL-23 by immune cells in heart infiltrates. Curiously, the medium inoculum showed the less favorable host response, which may indicate the existence of an ideal initial inoculum to help parasite evade host immune response (Borges et al., 2012).