*3.2.2. Cytokines and chemokines*

bites. The agent reaches the interior of macrophages when they phagocyte apoptotic bodies of previously infected neutrophils, where it can survive and multiply. This parasite escape

The main effector mechanism involved in protective immune response against *Leishmania* spp*.* is the activation of macrophages by IFN-γ and tumor necrosis factor alpha (TNF-α) stimulation, with consequent stimulation of nitric oxide synthesis, which is required for effective destruction of the pathogen and for controlling the spread of infection in dogs [81–83].

The major subpopulations of lymphocytes are CD4+ T cells (Th1, producing IFN-γ and TNFα; Th2, secreting IL-4, IL-5, and IL-13; and Th17, producing IL-17 and IL-22) and CD8+ T cells. Antigen-presenting cells submit *Leishmania* spp. antigens to CD4+ T cells via MHC class II, and because the agent is an intracellular parasite, there may be presentation via MHC class I with

The role of CD4+ T cells in the response to visceral leishmaniasis (VL) has not been fully elucidated. Research carried out so far points to a mixed response (Th1/Th2) during infection [84, 85]. It is reported, however, that control of infection depends on Th1 cells that activate macrophages, promoting elimination of intracellular parasites [86], whereas Th2 cells direct the immune system toward humoral response and negatively regulate cellular immunity,

CD8+ cells constitute a significant population in cellular immunity against canine visceral leishmaniasis (CVL), outnumbering CD4+ cells in the dermis [87]. They play an important role in resistance to infection. Guerra et al. [88] associated phenotypic changes with tissue parasitism in the spleen and skin of infected dogs. They noticed that the high frequency of CD8+ circulating lymphocytes is directly related to low splenic parasitism and that there is a negative correlation between CD8+ T cells with skin parasite density, indicating that this cell type relates

The regulatory role of FOXP3+ CD4+ T cells in canine VL has not been fully elucidated; however, reduction of Treg cell percentage in peripheral blood of infected dogs has been observed [89]. Silva et al. [90] reported increased production of IL-10 by splenic Treg cells of dogs with LV, along with decrease in the total number of T cells when compared to healthy dogs. The findings suggest that Treg cells are a major source of IL-10 in the spleen and participate in the modulation of immune response, while a small percentage of these cells in

T-cell exhaustion (CD4+ and CD8+ cells) in peripheral blood of dogs with LV, followed by reduction of the expression of cytokines (such as IFN-γ), was recently demonstrated. This phenomenon, called cell exhaustion, is mainly mediated by high expression of programmed death protein ("programmed cell death 1," PD-1) and may be related to the strong immuno-

infected dogs may be related to persistent immune activation [90].

mechanism is called "Trojan Horse" [80].

28 Canine Medicine - Recent Topics and Advanced Research

activation of CD8+ T cells as well [69].

promoting Th1 cell anergy [87].

to resistance against LVC [88].

**3.2. Acquired response**

*3.2.1. Lymphocytes*

Cytokine patterns for CVL have not been well established. Studies are inconclusive, so the pattern of immune response associated with resistance or susceptibility in infected animals is yet to be established.

One of the first studies on cytokine profiling in CVL was performed by Pinelli et al. [71], who observed high levels of IL-2 and tumor necrosis factor alpha (TNF-α) in asymptomatic dogs, compared with symptomatic ones, which suggests a role of these cytokines in resistance to *L. infantum* in dogs naturally or experimentally infected. Since then, much research has been done in order to elucidate the cytokine profiles found in various tissue compartments of infected dogs. They revealed contrasting cytokine profiles among different tissues, indicating that the immune response in LVC occurs in an organ-specific manner [71].

Profile of cytokines in peripheral blood mononuclear cells (PBMC) culture from asymptomatic dogs experimentally infected with *L. infantum* shows a predominantly Th1 response, mediated by expression of IL-2, IFN-γ, and IL-18 and very low IL-4 expression [95]. De Lima [96] dosed IL-6 and TNF-α in serum of symptomatic dogs naturally infected with *L. infantum* and uninfected control dogs. TNF-α was found in similar levels in dogs of both groups, yet IL-6 showed statistically higher levels in dogs with active VL than healthy dogs, thus demonstrating to be a good indicator for the disease [96].

After evaluating expression of cytokines in spleen cells from dogs naturally infected with *L. infantum*, Lage et al. suggested that CVL is characterized by a mixed response: with production of cytokines types I and II; involvement of IFN-y and IL-10 and a positive correlation between IL-10 levels and progression of parasitic load or clinical manifestations of the disease; and correlation between IFN-γ and increased parasitic intensity of the spleen [84]. In whole blood, the increase of IL-10 has been associated with detection of parasite DNA [97]. Do Nascimento et al., in accordance, state that in CVL there is increase in expression of pro-inflammatory cytokines IFN-γ and TNF-α in dogs with low splenic parasitism, while dogs with higher parasitic load show an increase in IL-10 [98].

Souza [99] states that asymptomatic dogs have low dermal parasitism and exhibit a mixed pattern of immune response, with simultaneous increase of type I (IFN-γ and TNF-α) and type II (IL-5 and IL-13) cytokines, but predominance of type I response. According to the author, increased and simultaneous expression of IFN-γ and TNF-α in the skin of infected dogs enables the speculation that these mediators are closely involved with protection mechanisms during CVL, since these cytokines increased in the skin of animals with the asymptomatic clinical form. Increased expression of IL-5 and IL-13 in the skin of healthy dogs and negative correlation of the latter with clinical disease progression were also observed. Furthermore, high simultaneous expression of IFN-γ and IL-13 was found in asymptomatic dogs, indicating the role of IL-13 in establishing milder clinical forms [99].

Regarding cytokine profile in the bone marrow, Quinnell et al. [94] reported that expression of mRNA for IL-10, IL-4, and IL-18 was not elevated in infected dogs. However, some infected dogs had detectable expression of mRNA for IL-4 significantly correlated with more severe clinical signs. Moreover, expression of mRNA for IL-13 was not detected either in control or in infected dogs, and unlike in human infection, immunosuppressive activity of IL-10 was not observed in CVL [94].

Dogs infected with *L. infantum* exhibit significant decrease in expression of mRNAs for IL-10, IL-17, TNF-α, IFN-γ, and iNOS in liver tissue. Deficiency in IL-17 mRNA expression was evident in the symptomatic dogs compared to the asymptomatic. Reduction in cytokine expression results in decreased iNOS expression and therefore higher parasite load. The increase in IL-17 expression in the liver of asymptomatic dogs and its correlation with elevated expression of iNOS indicates a protective role of that cytokine in canine infection by *L. infantum* [67]. However, Michelin et al. [100] reported increased TNF-α, IL-4, and IL-10 levels in the liver of infected dogs compared to healthy dogs. In addition, the association between TNF-α levels and an increase in parasitic load in the spleen suggests its importance in the evolution of the infection process [100].

Another subject lacking clarification is the participation of chemokines and their receptors in resistance or susceptibility to LVC. Knowledge surrounding the role of these modulators in response to *L. infantum* infection is critical, considering that the interaction between cytokines and chemokines may regulate the immune response against the parasite, activating and recruiting immune cells to areas of infection.

Menezes-Souza et al. [101] analyzed the expression of CCL2, CCl4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8 chemokines in the skin of 35 dogs naturally infected by *L. infantum*, comparing cutaneous parasitism and clinical manifestations of the disease. Increase of the parasitic load correlated with increased expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. On the other hand, CCL24 expression negatively correlated with parasitism [101].

After connecting clinical findings in naturally infected dogs with liver and spleen parasitism and expression levels for cytokines, chemokines, and their receptors, Albuquerque [67] showed that symptomatic dogs exhibit low expression of these modulators, alongside lower inflammatory response, and higher parasite load—primarily in the liver—than asymptomatic animals. CXCL10 was the only chemokine found at a much higher concentration in both the liver and the spleen of symptomatic animals. It also positively correlated with clinical score. The author indicates that expression profiles of hepatic and splenic chemokines and their receptors are essential for induction of correct cell inflammatory profile, as it has potential to contain the infection and the disease. Impaired cell migration facilitates replication of the parasite and development of CVL symptoms [67].

Understanding of the immune response in canine visceral leishmaniasis may reveal the factors involved with the onset and severity of clinical signs and the damage to host tissues. Additionally, it takes place as an indispensable tool for development of an effective vaccine.
