**3. Immunological parameters in the context of the diversity of retinal/retinochoroidal scar lesions from** *T. gondii* **seropositive patients**

*T. gondii* infection in both mice and humans is characterized by a host response with high levels of pro-inflammatory cytokines, such as interleukin 12 (IL-12), tumor necrosis factor (TNF)-α and interferon gamma (IFN- γ), all of which have been implicated in both the regulation of parasitic replication in the host as well as in the ocular pathology [11, 16, 21- 24]. The importance of the immune response of patients infected with *T. gondii* against the parasite has been recognized, together with other variables comprising the multifactorial nature of ocular toxoplasmosis. In this section, we present results from the *in vitro* parameters of the cellular immune response of *T. gondii* seropositive patients with and without ocular retinal/retinochoroidal scars and control groups of seronegative patients exposed to the same risk of infection, which involved the consumption of untreated water from wells or other natural water sources [1], against soluble antigens from *T. gondii* tachyzoite forms (STAg). Cytokines, chemokines, and isotypes of immunoglobulins have been evaluated in an effort to identify potential predictive factors for the development or prevention of ocular disease. All of the immunological parameters have been analyzed considering different groups of patients arranged according to the similarity in the retinal/retinochoroidal scar lesion group.

A pro-inflammatory specific T helper 1 (Th-1) oriented response is observed mainly in groups of patients presenting retinochoroidal scar healed from severe lesions, which suggests that the exacerbation of the immune response can be related to tissue damage, and its attenuation/regulation may be related to the development of minor retinal damages. The central role of IFN- seems to be important in both cases, namely, in exacerbated and in the regulated context of in vitro cellular immune response, suggesting that the cellular immune responses against *T. gondii* in the eye should be suitably tailored [11, 16, 21-23], as we shall see later. Other molecules and cells related to the regulation of secretion of IFN-γ include IL-13, chemokines, isotypes of immunoglobulins, NK cells and T CD8 lymphocytes in relation to the development or prevention of development of toxoplasmic ocular pathology have also been investigated [11, 16, 21, 22]. The immunological parameters studied have also made way for the election of candidate genes to be investigated in studies of genetic association with ocular toxoplasmosis, as we shall see in the next section.

However, we have evaluated many immunological parameters we have chosen three to better illustrate the profile of the cellular immune response as a function of the type of scar lesion presented by patients. The first is IFN-, the prototype of Th-1 response that has been shown to be of vital importance for inducing anti-*T. gondii* effectors mechanisms to control the parasite replication in the host [25]. The second, IL-13, is a Th-2 cytokine whose functions overlap considerably with those of IL-4, and it is important for the control of the Th-1 response but that has not been well studied in toxoplasmosis. The third chemokine, CXCL 10 (interferon gamma-induced protein 10- IP-10), is secreted in response to IFN stimulation. Recently, it was demonstrated in a murine model that treatment of chronically infected mice with anti-CXCL10 antibodies led to decreases in the numbers of CD3+, CD4+, and CD8+ T cells and the amount of IFN- mRNA expression in the retina and an increase in replicating parasites and ocular pathology, which provides evidence that the maintenance of the T-cell response and the control of *T. gondii* in the eye during chronic infection is dependent on CXCL10 [26]. Cytokines and chemokine measurements were carried out using supernatants collected from PBMC cultures stimulated with *T. gondii* antigens. The concentrations were determined by using the BD Cytometric Bead Array (CBA) human chemokine kit and Th1/Th2 cytokine kits and Human IL-13 Flex Set, according to the manufacturer's protocol (BD Pharmingen).

Immunological and Immunogenetic Parameters on the Diversity of Ocular Toxoplasmosis: Evidence to Support Morphological Criteria to Classify Retinal/Retinochoroidal Scar Lesions in Epidemiologic Surveys 157

scar lesion type n Mean Age (SE)1

producers. To calculate the frequency of high and low levels of production, an arbitrary cutoff value was established for each cytokine and chemokine based on the visual dispersion graphics, where it was possible to determine a dividing line that separated the secretion levels into two scattered clusters, one high and another low. The capability of producing low and high levels of IFN- is a phenotypic characteristic that is genetically associated with a single nucleotide polymorphism in the first intron of the human IFN- gene, as determined by Pravica and colleagues in 2000 [27]. The capability of producing low and high levels of IFN can be observed in other infectious disease [28, 29]. The levels secreted by each patient that fell above the cutoff values were considered high values, and the levels that fell below the cutoff values were considered low values. The median value of the two cytokines IFN- and IL-13 and the chemokine CXCL10 secretion was utilized to help to establish the exact numeric cutoff values to be used for each cytokine and chemokine: 29.58ng./mL, 194.12 pg/mL, and 113.72 ng/mL for IFN-, IL-13 and CXCL10, respectively. Those median values refer to patients who presented type C scar lesions. This group was chosen because it exhibited the best spreading profile, producing two very clearly separated clusters corresponding to levels of high and low

producers of the two cytokines and the chemokine that were evaluated.

1 SN2 9 29.6 (5.2) 2 SP3/NL4 21 28.8 (4.7) 3 SP/type A5-6 18 35.5 (6.0) 4 SP/type B5-6 21 27.5 (4.4) 5 SP/type C5 20 31.7 (5.1)

<sup>5</sup> *T. gondii*-seropositive individuals (SP) with retinochoroidal/retinal scars lesions categorized as class type A, type B or

6 In the group of type A scar lesions, individuals with single type A scar lesions (N=4) and multiple scars lesions type AB (N=5), ABC (N=7) and AC (N=2) were also included, totaling the 18 individuals shown in the table. In the group of type B scar lesions, individuals with single type B lesions (N=14) and individuals with multiple scars lesions type BC

**Table 4.** Groups of Individuals Involved in Immunological Studies According to *Toxoplasma gondii*

Figure 3 shows the frequency of IFN-, IL-13 and the chemokine CXCL 10 secretion, considering only patients who produced high levels of each cytokine, i.e., the high producers. The lowest frequency of high IFN- producers (43%) was observed in the group of patients with type B scar lesions even compared with patients presenting no lesions (SL) (52%), and the highest (61%) was observed in the group that presented type A scar lesions; 50% of the patients with type C lesions presented high levels of IFN- production. However, for IL-13 production, we observed that the lowest frequency of high producers was observed among patients with type A (28%) scar lesions, in comparison with patients who presented no lesions (SL) (29%). The highest production levels were observed among

Serology, Age and the Presence or Absence of Retinal/Retinochoroidal Scar Lesions.

Groups Toxoplasmosis serology and

<sup>3</sup> *T. gondii*-seropositive individuals (SP) without ocular lesions 4(NL)

(N=7) were included, totaling the 21 individuals shown in the table.

1 (SE) = standard error

type C.

*2 T. gondii*-seronegative individuals (SN)

Table 4 summarizes the individuals for which the specific immune response against *T. gondii* has been evaluated. The *T. gondii* serology and age range is provided. All the seronegative (SN) individuals, seropositive (SP) without ocular scar lesions (NL) and individuals with retinal or retinochoroidal scar lesions categorized as type A, type B or type C, as explained above, were sex- and age-matched among the groups. We observed that there was a similarity in terms of the profile of the *in vitro* immune response between patients who presented multiple scar type lesions and patients who presented single type A or B scar lesions, depending on which cytokine had been considered among the groups for comparison. Then immunological analysis, presented in Figure 3 and Figure 4, show the patients presenting multiple type scar lesions (AB, ABC, AC or BC) grouped in two different ways. This optional arrangement of patients, considering the multiple types of scar lesions, has helped us to propose three settings of immune responses that match the clinical presentation of ocular toxoplasmosis, which has been inferred in our studies by the morphological appearance of the retinal/retinochoroidal lesions. The reasoning behind the optional arrangements is related to the common presence of type B and type A scar lesions in two (AB and ABC) out of the four (AB, ABC, AC and BC) multiple type scar lesions. In Figure 3, they were grouped as follows: patients with AB, ABC and AC scar lesions are all included in the type A scar lesion group, which we considered the highest categorization in terms of tissue damage severity of scar lesion, and the patients presenting scar lesions of type BC were included in the group of type B scar lesions. The type C scar lesion group is composed of individuals presenting only type C scar lesions.

Concerning to the cytokine and chemokine production, we observed two levels of production that are termed as high or low levels. As a consequence, individuals producing low or high levels of cytokines or chemokines are termed high or low cytokine/chemokine producers. To calculate the frequency of high and low levels of production, an arbitrary cutoff value was established for each cytokine and chemokine based on the visual dispersion graphics, where it was possible to determine a dividing line that separated the secretion levels into two scattered clusters, one high and another low. The capability of producing low and high levels of IFN- is a phenotypic characteristic that is genetically associated with a single nucleotide polymorphism in the first intron of the human IFN- gene, as determined by Pravica and colleagues in 2000 [27]. The capability of producing low and high levels of IFN can be observed in other infectious disease [28, 29]. The levels secreted by each patient that fell above the cutoff values were considered high values, and the levels that fell below the cutoff values were considered low values. The median value of the two cytokines IFN- and IL-13 and the chemokine CXCL10 secretion was utilized to help to establish the exact numeric cutoff values to be used for each cytokine and chemokine: 29.58ng./mL, 194.12 pg/mL, and 113.72 ng/mL for IFN-, IL-13 and CXCL10, respectively. Those median values refer to patients who presented type C scar lesions. This group was chosen because it exhibited the best spreading profile, producing two very clearly separated clusters corresponding to levels of high and low producers of the two cytokines and the chemokine that were evaluated.


1 (SE) = standard error

156 Toxoplasmosis – Recent Advances

manufacturer's protocol (BD Pharmingen).

composed of individuals presenting only type C scar lesions.

However, we have evaluated many immunological parameters we have chosen three to better illustrate the profile of the cellular immune response as a function of the type of scar lesion presented by patients. The first is IFN-, the prototype of Th-1 response that has been shown to be of vital importance for inducing anti-*T. gondii* effectors mechanisms to control the parasite replication in the host [25]. The second, IL-13, is a Th-2 cytokine whose functions overlap considerably with those of IL-4, and it is important for the control of the Th-1 response but that has not been well studied in toxoplasmosis. The third chemokine, CXCL 10 (interferon gamma-induced protein 10- IP-10), is secreted in response to IFN stimulation. Recently, it was demonstrated in a murine model that treatment of chronically infected mice with anti-CXCL10 antibodies led to decreases in the numbers of CD3+, CD4+, and CD8+ T cells and the amount of IFN- mRNA expression in the retina and an increase in replicating parasites and ocular pathology, which provides evidence that the maintenance of the T-cell response and the control of *T. gondii* in the eye during chronic infection is dependent on CXCL10 [26]. Cytokines and chemokine measurements were carried out using supernatants collected from PBMC cultures stimulated with *T. gondii* antigens. The concentrations were determined by using the BD Cytometric Bead Array (CBA) human chemokine kit and Th1/Th2 cytokine kits and Human IL-13 Flex Set, according to the

Table 4 summarizes the individuals for which the specific immune response against *T. gondii* has been evaluated. The *T. gondii* serology and age range is provided. All the seronegative (SN) individuals, seropositive (SP) without ocular scar lesions (NL) and individuals with retinal or retinochoroidal scar lesions categorized as type A, type B or type C, as explained above, were sex- and age-matched among the groups. We observed that there was a similarity in terms of the profile of the *in vitro* immune response between patients who presented multiple scar type lesions and patients who presented single type A or B scar lesions, depending on which cytokine had been considered among the groups for comparison. Then immunological analysis, presented in Figure 3 and Figure 4, show the patients presenting multiple type scar lesions (AB, ABC, AC or BC) grouped in two different ways. This optional arrangement of patients, considering the multiple types of scar lesions, has helped us to propose three settings of immune responses that match the clinical presentation of ocular toxoplasmosis, which has been inferred in our studies by the morphological appearance of the retinal/retinochoroidal lesions. The reasoning behind the optional arrangements is related to the common presence of type B and type A scar lesions in two (AB and ABC) out of the four (AB, ABC, AC and BC) multiple type scar lesions. In Figure 3, they were grouped as follows: patients with AB, ABC and AC scar lesions are all included in the type A scar lesion group, which we considered the highest categorization in terms of tissue damage severity of scar lesion, and the patients presenting scar lesions of type BC were included in the group of type B scar lesions. The type C scar lesion group is

Concerning to the cytokine and chemokine production, we observed two levels of production that are termed as high or low levels. As a consequence, individuals producing low or high levels of cytokines or chemokines are termed high or low cytokine/chemokine *2 T. gondii*-seronegative individuals (SN)

<sup>3</sup> *T. gondii*-seropositive individuals (SP) without ocular lesions 4 (NL)

<sup>5</sup> *T. gondii*-seropositive individuals (SP) with retinochoroidal/retinal scars lesions categorized as class type A, type B or type C.

6 In the group of type A scar lesions, individuals with single type A scar lesions (N=4) and multiple scars lesions type AB (N=5), ABC (N=7) and AC (N=2) were also included, totaling the 18 individuals shown in the table. In the group of type B scar lesions, individuals with single type B lesions (N=14) and individuals with multiple scars lesions type BC (N=7) were included, totaling the 21 individuals shown in the table.

**Table 4.** Groups of Individuals Involved in Immunological Studies According to *Toxoplasma gondii* Serology, Age and the Presence or Absence of Retinal/Retinochoroidal Scar Lesions.

Figure 3 shows the frequency of IFN-, IL-13 and the chemokine CXCL 10 secretion, considering only patients who produced high levels of each cytokine, i.e., the high producers. The lowest frequency of high IFN- producers (43%) was observed in the group of patients with type B scar lesions even compared with patients presenting no lesions (SL) (52%), and the highest (61%) was observed in the group that presented type A scar lesions; 50% of the patients with type C lesions presented high levels of IFN- production. However, for IL-13 production, we observed that the lowest frequency of high producers was observed among patients with type A (28%) scar lesions, in comparison with patients who presented no lesions (SL) (29%). The highest production levels were observed among patients who presented type C scar lesions (50%); 30% of the patients with type B scar lesions presented high levels of IL-13 production. Curiously, for the CXCL 10 chemokine, which is inducible in response to IFN-, the highest frequency of high producers was observed among patients with type C scar lesions (53%), and the lowest frequency of high producers was observed among patients with type B scar lesions (21%), which is comparable to the frequency of high production observed in patients without ocular lesions (SL) (23%); 38% of patients with type A scar lesions were high producers of CXCL10. These data suggest that CXCL10 in humans can have the same role regarding *T. gondii* infection that it plays in mice: to control the numbers of CD3+, CD4+, and CD8+ T cells and the amount of IFN- mRNA expression in the retina and, in consequence, the replication of parasites in the eye during the chronic phase of *T. gondii* infection [26]. In addition, it is evident that patients who present type C scar lesions secrete high levels of IL-13, a cytokine that can control the levels of pro-inflammatory cytokines without affecting IFN- secretion within the eye, as shown in experimental models. The role of IL-13 was shown to inhibit pro-inflammatory cytokines, with the exception of IFN-, within the eye in a model of endotoxin-induced uveitis (EIU) in the Lewis rats. Intraocular injection of IL-13 significantly inhibited the production of pro-inflammatory cytokines and resulted in less intense ocular inflammation without down-regulating the levels of local IFN- [30]. In addition, the induced auto-immune uveitis with human retinal S-antigen in monkeys was treated with human recombinant IL-13 [31]. The injection of IL-13 significantly inhibited the inflammation in the eyes where the disease was present when the treatment was initiated. The beneficial effect of IL-13 extended into the 4-week follow-up period; however, after cessation of therapy, there was a progressive increase of inflammation in the IL-13 treated group. Nevertheless, the authors concluded that attention should be paid to the promising modality of treatment for severe uveitis with IL-13 [31]. The profile of the immune response against *T. gondii* exhibited by patients who presented type C scar lesions is suggestive of a favorable inflammatory environment within the eye for the maintenance of a controlled response to prevent both parasite growth and tissue damage, which could be caused by the parasite growth itself and/or may be a consequence of an exacerbated immune response against the parasite. In this context, it is important to highlight the fundamental importance that has been demonstrated for the role of IFN- in toxoplasmosis [25].

Immunological and Immunogenetic Parameters on the Diversity of Ocular Toxoplasmosis: Evidence to Support Morphological Criteria to Classify Retinal/Retinochoroidal Scar Lesions in Epidemiologic Surveys 159

**Figure 3.** Frequency of high IFN-γ IL-13 and CXCL10 (IP-10) producers, which were grouped according to the type of scar lesion, as shown in Table 4. The frequency of non-infected seronegative individuals (SN) is not shown because in none of them PBMC produced high levels of IFN-gamma, IL-13 and or CXCL10 (IP-10) under stimulation with soluble *T. gondii* antigens (STAg). The exact Fisher's test was used to compare the groups in terms of the differences in the frequencies of high cytokines/chemokine producers. Significances were found at the levels of \* p< 0.05, \*\* p< 0.01 and \*\*\* p< 0.001. Significant differences were found between type A and type B scar lesions for IFN-γ production (\* p< 0.05), between type C and all the other types of scars lesions regarding IL-13 secretion, and between type C and all the other types of scars lesions groups for CXCL10 (IP-10). The data regarding PBMC IFN-, IL-13 and CXCL10 from non-infected (SN) individuals in response to *T. gondii* soluble antigens is not shown because none of those patients

Based on fundoscopic examinations, type C scar lesions seem to be areas of RPE hyperplasia or atrophy. However, they must to be better characterized by new high-resolution crosssectional imaging of the retinal tissues (such as using spectral domain optical coherence tomography) in order to better clarify their structural changes in the retinal layers. As mentioned earlier some aspects showed here led us to accept the relationship between type C scar lesions and *T. gondii* infection one of them is the profile of cellular specific immune response of patients who present only type C scar lesions, which is concomitantly abundant for IFN-, IL-13 and CXCL10. The reproduction of this type of scar lesion in experimental models would be of value to better understand their meaning in ocular toxoplasmosis. Because we have been working in a highly endemic area to toxoplasmosis and the majority

produced high levels of those cytokines or chemokine.

IFN- can induce tryptophan degradation, which is critical to the parasite's survival [32]. The effects of interferon on multiplication of *T. gondii* in *in vitro* systems seem to be dependent on cell type, and a diversity of molecular mechanisms is evident depending on the cell type or system. It has been shown in neuronal tissues and cells that nitric oxide (NO) production is an important factor for parasite growth inhibition [33]. However, parasite growth inhibition was found to be independent of a nitric oxide-mediated or tryptophan starvation mechanism [34]. The control of parasite interconversion between tachyzoites and bradyzoites in the eye is also fundamentally dependent on IFN- [35]. Furthermore, in primary cultures of human retinal pigment cells (HRPE), *T. gondii* replication was inhibited by the induction of indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan to kynurenine. However, nitric oxide production was not detected in this system [36].

Immunological and Immunogenetic Parameters on the Diversity of Ocular Toxoplasmosis: Evidence to Support Morphological Criteria to Classify Retinal/Retinochoroidal Scar Lesions in Epidemiologic Surveys 159

158 Toxoplasmosis – Recent Advances

toxoplasmosis [25].

patients who presented type C scar lesions (50%); 30% of the patients with type B scar lesions presented high levels of IL-13 production. Curiously, for the CXCL 10 chemokine, which is inducible in response to IFN-, the highest frequency of high producers was observed among patients with type C scar lesions (53%), and the lowest frequency of high producers was observed among patients with type B scar lesions (21%), which is comparable to the frequency of high production observed in patients without ocular lesions (SL) (23%); 38% of patients with type A scar lesions were high producers of CXCL10. These data suggest that CXCL10 in humans can have the same role regarding *T. gondii* infection that it plays in mice: to control the numbers of CD3+, CD4+, and CD8+ T cells and the amount of IFN- mRNA expression in the retina and, in consequence, the replication of parasites in the eye during the chronic phase of *T. gondii* infection [26]. In addition, it is evident that patients who present type C scar lesions secrete high levels of IL-13, a cytokine that can control the levels of pro-inflammatory cytokines without affecting IFN- secretion within the eye, as shown in experimental models. The role of IL-13 was shown to inhibit pro-inflammatory cytokines, with the exception of IFN-, within the eye in a model of endotoxin-induced uveitis (EIU) in the Lewis rats. Intraocular injection of IL-13 significantly inhibited the production of pro-inflammatory cytokines and resulted in less intense ocular inflammation without down-regulating the levels of local IFN- [30]. In addition, the induced auto-immune uveitis with human retinal S-antigen in monkeys was treated with human recombinant IL-13 [31]. The injection of IL-13 significantly inhibited the inflammation in the eyes where the disease was present when the treatment was initiated. The beneficial effect of IL-13 extended into the 4-week follow-up period; however, after cessation of therapy, there was a progressive increase of inflammation in the IL-13 treated group. Nevertheless, the authors concluded that attention should be paid to the promising modality of treatment for severe uveitis with IL-13 [31]. The profile of the immune response against *T. gondii* exhibited by patients who presented type C scar lesions is suggestive of a favorable inflammatory environment within the eye for the maintenance of a controlled response to prevent both parasite growth and tissue damage, which could be caused by the parasite growth itself and/or may be a consequence of an exacerbated immune response against the parasite. In this context, it is important to highlight the fundamental importance that has been demonstrated for the role of IFN- in

IFN- can induce tryptophan degradation, which is critical to the parasite's survival [32]. The effects of interferon on multiplication of *T. gondii* in *in vitro* systems seem to be dependent on cell type, and a diversity of molecular mechanisms is evident depending on the cell type or system. It has been shown in neuronal tissues and cells that nitric oxide (NO) production is an important factor for parasite growth inhibition [33]. However, parasite growth inhibition was found to be independent of a nitric oxide-mediated or tryptophan starvation mechanism [34]. The control of parasite interconversion between tachyzoites and bradyzoites in the eye is also fundamentally dependent on IFN- [35]. Furthermore, in primary cultures of human retinal pigment cells (HRPE), *T. gondii* replication was inhibited by the induction of indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan to

kynurenine. However, nitric oxide production was not detected in this system [36].

**Figure 3.** Frequency of high IFN-γ IL-13 and CXCL10 (IP-10) producers, which were grouped according to the type of scar lesion, as shown in Table 4. The frequency of non-infected seronegative individuals (SN) is not shown because in none of them PBMC produced high levels of IFN-gamma, IL-13 and or CXCL10 (IP-10) under stimulation with soluble *T. gondii* antigens (STAg). The exact Fisher's test was used to compare the groups in terms of the differences in the frequencies of high cytokines/chemokine producers. Significances were found at the levels of \* p< 0.05, \*\* p< 0.01 and \*\*\* p< 0.001. Significant differences were found between type A and type B scar lesions for IFN-γ production (\* p< 0.05), between type C and all the other types of scars lesions regarding IL-13 secretion, and between type C and all the other types of scars lesions groups for CXCL10 (IP-10). The data regarding PBMC IFN-, IL-13 and CXCL10 from non-infected (SN) individuals in response to *T. gondii* soluble antigens is not shown because none of those patients produced high levels of those cytokines or chemokine.

Based on fundoscopic examinations, type C scar lesions seem to be areas of RPE hyperplasia or atrophy. However, they must to be better characterized by new high-resolution crosssectional imaging of the retinal tissues (such as using spectral domain optical coherence tomography) in order to better clarify their structural changes in the retinal layers. As mentioned earlier some aspects showed here led us to accept the relationship between type C scar lesions and *T. gondii* infection one of them is the profile of cellular specific immune response of patients who present only type C scar lesions, which is concomitantly abundant for IFN-, IL-13 and CXCL10. The reproduction of this type of scar lesion in experimental models would be of value to better understand their meaning in ocular toxoplasmosis. Because we have been working in a highly endemic area to toxoplasmosis and the majority of the population is *T. gondii* seropositive, we cannot rule out the possibility of an association between the type C scar lesions and other infectious or non-infectious conditions whose natures could be genetically predisposed and prevalent in the Campos population.

Immunological and Immunogenetic Parameters on the Diversity of Ocular Toxoplasmosis: Evidence to Support Morphological Criteria to Classify Retinal/Retinochoroidal Scar Lesions in Epidemiologic Surveys 161

17A production by PBMC under *T. gondii* antigenic stimulation in patients with chronic or active ocular toxoplasmosis [37]. This fact could explain the lower levels of IL-13, IFN- and CXCL10 observed for patients who present type B and multiple type scar lesions. However, we have not analyzed patients considering their type of scar lesion groups for the mentioned genetic association study due to sample size restrictions, and the genetic association found for *NOD2* refers to the presence of ocular scar lesions without precisely

**Figure 4.** A Diagram representing retinal/retinochoroidal toxoplasmic scar lesions, as a function of three different settings of cellular immune response. The platonic solid octahedron in the center is suitable for representing the diversity of scar lesions as a function of the three settings of immune response. On the two edges are the type A (at the top) and type C (at the bottom) scar lesions. In the four central vertices of octahedron are represented the type B scar lesion and the multiple type scar lesions AB, AC and BC, and in the center is a multiple type scar lesion of type ABC. The dotted and solid arrows denote the possible evolution of one type of scar lesion toward another type of lesion of greater severity. Not all possibilities of

IL-13 and CXCL10 (IP-10) high-responders that are shown in the graphics at right define the three settings of the immune response shown at left. Each of the three settings correlates with each of the three groups: with the scar lesions of the type A at the edge, with the scar lesion of type C at the bottom, or with the type B scar lesion plus all the multiple type lesions. The blue arrows shown in the settings denote the levels of cytokines and chemokine, as shown in the figure. The scar lesion types are represented by colored circles,

The data regarding PBMC IFN-, IL-13 and CXCL10 from non-infected (SN) individuals in response to *T. gondii* soluble antigens is not shown in Figure 3 and Figure 4 because none of those patients produced high levels of the cytokines or chemokine in question. In conclusion, elements from the cellular immune response, evaluated in PBMC cultures of population-based studies evidence that the morphological aspects of retinal/retinochoroidal

identifying what type of scar lesion it could be.

evolution are shown in the picture. The frequencies of IFN-,

as explained in the figure.

As stated previously, patients were grouped in an optional way. Those presenting only type A scar lesions comprised a distinct group, taking into account the possibility that in patients presenting only type A scar lesions, the course of the immune response could be different from that which occurred in patients who presented multiple type scar lesions. There are rational and intuitive aspects to this arrangement that are related to various factors, like the tendency for the production of higher IFN- levels of in PBMC cultures of patients who present only type A scar lesions in response to parasitic antigens and the clinical observation that some patients can present type A scar lesions soon after an episode of acute ocular toxoplasmosis without a previous history of ocular toxoplasmosis. However, some patients present a slower evolution, from mild to severe scar lesions, as described by Silveira and associates in a well documented report [13]. As previously stated in this chapter, this observation concerns the evolution of hyperpigmented "atypical" toxoplasmic retinal scar lesions (similar in appearance to the type B toxoplasmic scar lesions that we have described) evolving to "typical" toxoplasmic retinochoroidal lesions (similar in appearance to the type A toxoplasmic scar lesions that we have described). Then for the analysis of the immune response as a function of the clinical presentation of ocular toxoplasmosis inferred by the morphological appearance of the retinal/retinochoroidal scar lesions, the patients were optionally arranged into three groups as follows: i) patients presenting only type A scar lesions; ii) patients presenting only type B scar lesions plus patients presenting all the multiple type scar lesions (AB + ABC + AC + BC); and iii) patients presenting only type C scar lesions. Figure 4 summarizes the *in vitro* parameters of the cellular immune response against *T. gondii* antigens by PBMC of patients in the context of the retinal/retinochoroidal lesions considering the three groups of type A, type C and type B plus the multiple type scar lesions. Similarly, as shown in Figure 3, considering the profiles of the frequency of high IFN-, IL-13 and CXCL10 production by PBMC of patients stimulated with *T. gondii* soluble antigens (STAg), we identified three possible settings of the immune response that can be associated with the type of scar lesion. As shown in Figure 4 the Platonic solid octahedron is suitable for representing the diversity of scar lesions as a function of the three settings of immune responses. On the two edges are the polar scar lesions, type A and type C. In the top is the type A scar, which is caused by the most severe type of lesion, and in the bottom is the type C scar that likely resulted from a less severe lesions type. Both types of scar lesions respectively correlate with the setting of an exacerbated and an adequately regulated Th-1 response. In the four central vertices are type B scar lesions and the multiple type scar lesions AB, AC and BC, and in the center of the octahedron is the multiple type scar lesion ABC, all of which correlate with the setting of an immune response characterized by lower levels of Th-1 (IFN- and CXCL10) and Th-2 (IL-13) compared with type A and C scar lesions. Taking into account this fact, it is possible that other T cells subtypes, such as Th-17, for instance, can be important in this setting. Reinforcing this supposition is the fact that we have observed recent associations of ocular toxoplasmosis and polymorphisms in the *NOD2* (nucleotide-binding oligomerization domain-containing protein 2) gene and increased IL-

17A production by PBMC under *T. gondii* antigenic stimulation in patients with chronic or active ocular toxoplasmosis [37]. This fact could explain the lower levels of IL-13, IFN- and CXCL10 observed for patients who present type B and multiple type scar lesions. However, we have not analyzed patients considering their type of scar lesion groups for the mentioned genetic association study due to sample size restrictions, and the genetic association found for *NOD2* refers to the presence of ocular scar lesions without precisely identifying what type of scar lesion it could be.

160 Toxoplasmosis – Recent Advances

of the population is *T. gondii* seropositive, we cannot rule out the possibility of an association between the type C scar lesions and other infectious or non-infectious conditions whose natures could be genetically predisposed and prevalent in the Campos population.

As stated previously, patients were grouped in an optional way. Those presenting only type A scar lesions comprised a distinct group, taking into account the possibility that in patients presenting only type A scar lesions, the course of the immune response could be different from that which occurred in patients who presented multiple type scar lesions. There are rational and intuitive aspects to this arrangement that are related to various factors, like the tendency for the production of higher IFN- levels of in PBMC cultures of patients who present only type A scar lesions in response to parasitic antigens and the clinical observation that some patients can present type A scar lesions soon after an episode of acute ocular toxoplasmosis without a previous history of ocular toxoplasmosis. However, some patients present a slower evolution, from mild to severe scar lesions, as described by Silveira and associates in a well documented report [13]. As previously stated in this chapter, this observation concerns the evolution of hyperpigmented "atypical" toxoplasmic retinal scar lesions (similar in appearance to the type B toxoplasmic scar lesions that we have described) evolving to "typical" toxoplasmic retinochoroidal lesions (similar in appearance to the type A toxoplasmic scar lesions that we have described). Then for the analysis of the immune response as a function of the clinical presentation of ocular toxoplasmosis inferred by the morphological appearance of the retinal/retinochoroidal scar lesions, the patients were optionally arranged into three groups as follows: i) patients presenting only type A scar lesions; ii) patients presenting only type B scar lesions plus patients presenting all the multiple type scar lesions (AB + ABC + AC + BC); and iii) patients presenting only type C scar lesions. Figure 4 summarizes the *in vitro* parameters of the cellular immune response against *T. gondii* antigens by PBMC of patients in the context of the retinal/retinochoroidal lesions considering the three groups of type A, type C and type B plus the multiple type scar lesions. Similarly, as shown in Figure 3, considering the profiles of the frequency of high IFN-, IL-13 and CXCL10 production by PBMC of patients stimulated with *T. gondii* soluble antigens (STAg), we identified three possible settings of the immune response that can be associated with the type of scar lesion. As shown in Figure 4 the Platonic solid octahedron is suitable for representing the diversity of scar lesions as a function of the three settings of immune responses. On the two edges are the polar scar lesions, type A and type C. In the top is the type A scar, which is caused by the most severe type of lesion, and in the bottom is the type C scar that likely resulted from a less severe lesions type. Both types of scar lesions respectively correlate with the setting of an exacerbated and an adequately regulated Th-1 response. In the four central vertices are type B scar lesions and the multiple type scar lesions AB, AC and BC, and in the center of the octahedron is the multiple type scar lesion ABC, all of which correlate with the setting of an immune response characterized by lower levels of Th-1 (IFN- and CXCL10) and Th-2 (IL-13) compared with type A and C scar lesions. Taking into account this fact, it is possible that other T cells subtypes, such as Th-17, for instance, can be important in this setting. Reinforcing this supposition is the fact that we have observed recent associations of ocular toxoplasmosis and polymorphisms in the *NOD2* (nucleotide-binding oligomerization domain-containing protein 2) gene and increased IL-

**Figure 4.** A Diagram representing retinal/retinochoroidal toxoplasmic scar lesions, as a function of three different settings of cellular immune response. The platonic solid octahedron in the center is suitable for representing the diversity of scar lesions as a function of the three settings of immune response. On the two edges are the type A (at the top) and type C (at the bottom) scar lesions. In the four central vertices of octahedron are represented the type B scar lesion and the multiple type scar lesions AB, AC and BC, and in the center is a multiple type scar lesion of type ABC. The dotted and solid arrows denote the possible evolution of one type of scar lesion toward another type of lesion of greater severity. Not all possibilities of evolution are shown in the picture. The frequencies of IFN-,

IL-13 and CXCL10 (IP-10) high-responders that are shown in the graphics at right define the three settings of the immune response shown at left. Each of the three settings correlates with each of the three groups: with the scar lesions of the type A at the edge, with the scar lesion of type C at the bottom, or with the type B scar lesion plus all the multiple type lesions. The blue arrows shown in the settings denote the levels of cytokines and chemokine, as shown in the figure. The scar lesion types are represented by colored circles, as explained in the figure.

The data regarding PBMC IFN-, IL-13 and CXCL10 from non-infected (SN) individuals in response to *T. gondii* soluble antigens is not shown in Figure 3 and Figure 4 because none of those patients produced high levels of the cytokines or chemokine in question. In conclusion, elements from the cellular immune response, evaluated in PBMC cultures of population-based studies evidence that the morphological aspects of retinal/retinochoroidal scar lesions can be associated with three settings of immune response in areas of high prevalence of ocular toxoplasmosis. The three settings are i) a Th-1 prominent response with high levels of IFN-, moderate to low levels of CXCL10 and low levels of IL-13, which relate to single type A scar lesions that are healed from the most severe toxoplasmic ocular lesions; ii) a cellular response with moderate to low levels of IFN- and IL-13 and low levels of CXCL10, which relate to multiple type and type B scar lesions; and iii) a sharply regulated Th-1 response with moderate to high levels of CXCL 10 and IL-13 and moderate levels of IFN-, which relate to type C scar lesions and could protect against tissue damage due to parasite replication within the eye.

Immunological and Immunogenetic Parameters on the Diversity of Ocular Toxoplasmosis: Evidence to Support Morphological Criteria to Classify Retinal/Retinochoroidal Scar Lesions in Epidemiologic Surveys 163

experts considered the retinal lesions in *T. gondii*-positive patients more frequently "consistent with the diagnosis of ocular toxoplasmosis" (P = .010 and P = 0.011). There was a

We cannot rule out the fact that none of the patients have been considered as cases of ocular toxoplasmosis in the Campos dos Goytacazes surveys or in surveys from other parts of Brazil, as scar lesions left from Rubella virus infection as opposed to *T. gondii* ocular infection were present in their eyes. However, we have to take into account that Rubella was a highly prevalent virus worldwide, including in Brazil, and it is probable that if ocular lesions left by Rubella infection were as frequent as in toxoplasmosis, such lesions would already have been described as a causative entity of uveitis with epidemiologic importance. Rubella virus-caused lesions currently no longer occur due to the vaccination program against Rubella virus that was undertaken in many countries, including in Brazil where it was introduced 15 years ago. Hence, despite the clinical relevance of the similarity between the Rubella virus- caused lesions and toxoplasma retinal/retinochoroidal scar lesions, we believe that from an epidemiologic perspective, this similarity is not relevant. However, these data reinforce that the search for parameters, other than the morphological and serological, for classifying retinal/retinochoroidal scar lesions presumably caused by *T. gondii* infections should be pursued. Parameters of cellular specific immune response or the genotyping of candidate genes with the potential to differentiate between infectious agents

substantial agreement between the four experts (Fleiss' Kappa = 0.623) [38].

that produce similar ocular lesions could be of help for disease management.

seronegative to *T. gondii* and positive to *A. lumbricoides* in our surveys.

We have reported recently in Campos that the host immune response to *T. gondii* and *Ascaris lumbricoides* evidence co-immune modulation properties that can influence the outcome of both infections. One of the most impressive aspect of the immune response of co-infected individuals is the prominent specific secretion of IL-13 against *Ascaris* and *T. gondii* antigens by PBMC of patients who present type C scar lesions in addition to middle to high secretion levels of IFN- [11]. This aspect of the immune response seems to be important for the control of the parasite retinal replication and most likely for the maintenance of an equilibrated *T. gondii* load and the interconversion of tachyzoites and bradyzoites in the eye tissues*. T. gondii* and *A. lumbricoides* are both parasites that infect hosts orally; however, they elicit polar type I or type II host responses, respectively. Because both parasites are endemic in tropical areas, it is likely that co-infections with these organisms have been common throughout human evolution. If this is the case, then the host immune response mounted against both parasites may have adapted to permit such co-parasitism. The possibility of *A. lumbricoides* to produce some type of ocular scar lesion in humans seems not to be of epidemiologic importance, as we have not detected ocular scar lesions in patients

The recent adoption of massive anti-helminthic treatments for people living in poor communities as a measure of public health policy has made difficult the research on individuals co-infected with *A. lumbricoides* and *T. gondii* in Campos dos Goytacazes as well as in other parts of Brazil. For this reason, we have begun to work with an experimental model of co-infection with *T. gondii* and *Heligmosomoides polygyrus*, which has been shown as
