**4. Ocular toxoplasmosis in association with other infectious diseases: Could this impact the clinical presentations of toxoplasmic scar lesions in population-based surveys?**

The interaction with other pathogens co-infecting *T. gondii* seropositive patients will depend on their exposure to such eventually co-prevalent parasites in the same area and on the type of immune response driven against them. The disease manifestation of *T. gondii* infected patients who become infected with HIV exemplify well this type of interaction and modification on the clinical presentation of ocular toxoplasmosis in the face of concomitant infections [3].

The scar lesions left by healed uveitis caused by *T. gondii* infections result from the previous process of uveal inflammation, which they have in common with other infectious and noninfectious agents that also cause uveitis, the possibility to compromise the iris, the cilliary body, the choroid adjacent structures of the eye including the vitreous, the retina and the optic disc. In this context, the thesis dedicated to this specific issue entitled "Infectious uveitis new developments in etiology and pathogenesis" from De Visser [38] underlines the necessity in clinical practice to have the support of laboratory data for the confirmation of a suspected diagnosis, as similar clinical features may be caused by different pathogens. The prompt identification of infectious uveitis entities is of vital importance for treatment regimens and visual prognoses of intraocular infections, and differ completely from noninfectious disorders treatments.

The thesis also reports on the similarity between retinal/retinochoroidal scar lesions left by Rubella eye infection and those left by ocular toxoplasmosis in a well-documented retrospective study. The clinical presentations of the focal retinal scars in 11 patients with intraocular proof of Rubella virus and in 17 patients with intraocular proof of *T. gondii* infection are compared. Photographic and angiographic records of the 28 patients were masked for identification and for infectious agent and were evaluated by four specialists in the field of ocular toxoplasmosis. It is reported that no differences were observed between the retinochoroidal scar lesions in Rubella virus-positive and *T. gondii*-positive patients. Retinochoroidal scar lesions were considered consistent with the diagnosis of ocular toxoplasmosis in 55% of Rubella virus-positive patients and in 88% of *T. gondii*-positive patients by at least three out of the four experts. According to the author of the thesis, two 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 substantial agreement between the four experts (Fleiss' Kappa = 0.623) [38].

162 Toxoplasmosis – Recent Advances

parasite replication within the eye.

**population-based surveys?** 

infectious disorders treatments.

infections [3].

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

**4. Ocular toxoplasmosis in association with other infectious diseases:** 

**Could this impact the clinical presentations of toxoplasmic scar lesions in** 

The interaction with other pathogens co-infecting *T. gondii* seropositive patients will depend on their exposure to such eventually co-prevalent parasites in the same area and on the type of immune response driven against them. The disease manifestation of *T. gondii* infected patients who become infected with HIV exemplify well this type of interaction and modification on the clinical presentation of ocular toxoplasmosis in the face of concomitant

The scar lesions left by healed uveitis caused by *T. gondii* infections result from the previous process of uveal inflammation, which they have in common with other infectious and noninfectious agents that also cause uveitis, the possibility to compromise the iris, the cilliary body, the choroid adjacent structures of the eye including the vitreous, the retina and the optic disc. In this context, the thesis dedicated to this specific issue entitled "Infectious uveitis new developments in etiology and pathogenesis" from De Visser [38] underlines the necessity in clinical practice to have the support of laboratory data for the confirmation of a suspected diagnosis, as similar clinical features may be caused by different pathogens. The prompt identification of infectious uveitis entities is of vital importance for treatment regimens and visual prognoses of intraocular infections, and differ completely from non-

The thesis also reports on the similarity between retinal/retinochoroidal scar lesions left by Rubella eye infection and those left by ocular toxoplasmosis in a well-documented retrospective study. The clinical presentations of the focal retinal scars in 11 patients with intraocular proof of Rubella virus and in 17 patients with intraocular proof of *T. gondii* infection are compared. Photographic and angiographic records of the 28 patients were masked for identification and for infectious agent and were evaluated by four specialists in the field of ocular toxoplasmosis. It is reported that no differences were observed between the retinochoroidal scar lesions in Rubella virus-positive and *T. gondii*-positive patients. Retinochoroidal scar lesions were considered consistent with the diagnosis of ocular toxoplasmosis in 55% of Rubella virus-positive patients and in 88% of *T. gondii*-positive patients by at least three out of the four experts. According to the author of the thesis, two 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 that produce similar ocular lesions could be of help for disease management.

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

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 valid model for studying parasites that evoke polar Th-1 and Th-2 in mice. *H. polygyrus* is a gastrointestinal worm, a natural parasite to mice, which evokes a polarized Th2 response in the host and blocks the type 1 immune response [39, 40]. It has been shown that a previous infection with the helminth can inhibit the development of CD8 T-cell immune response against *T. gondii*, thus compromising long-term protection against a protozoan parasite [41], which illustrates the adequacy of the model for studying the interaction between helminthic and *T. gondii* infections.

Immunological and Immunogenetic Parameters on the Diversity of Ocular Toxoplasmosis: Evidence to Support Morphological Criteria to Classify Retinal/Retinochoroidal Scar Lesions in Epidemiologic Surveys 165

Polymorphisms at the *P2RX7* have been investigated in a cooperative immunogenetic study involving patients from United State and Campos dos Goytacazes in Brazil. The studies comprised 149 child in 149 child/parent trios from North America and 60 families with 68 affected with postnatal ocular toxoplasmosis offspring in Brazil [18]. For the United states casuistic, an association between the derived C(+)G(−) allele and a second synonymous variant rs1621388 in linkage disequilibrium with it; and clinical signs of disease *per se.* were observed . Analysis of clinical sub-groups showed associations with retinal disease and brain calcifications (OR=3.0 to 4.25; 0.004<P<0.009). The association with toxoplasmic retinochoroiditis was replicated in a family-based study from Campos dos Goytacazes (60 families; 68 affected offspring), where the ancestral T(+) allele (f= 0.296) at SNP rs1718119 which contains the Ala-348 to Thr polymorphism was strongly protective (OR= 0.27; 95% CI: 0.09–0.80) [18]. This last association at SNP rs1718119 was recently replicated in a casecontrol study with 361 non-related individuals. The study confirmed the protective association of the T(+) allele (f= 0.296) (OR =0,3; 95% CI: 0,15-0,59 chi-square value = 13,53 *p*

corrected for multiple comparisons = 0.0132) for patients with type B scar lesions [49]

can contribute to retinal/retinochoroidal damage.

The SNPs at the *P2RX7* may be associated with a gain or loss of function of the receptor. Data from Stokes and colleagues [50] showed the gain of function for P2X7 haplotypes carrying rs1718119 SNP, which matches our recent case-control data that confirmed a strong protection due to the ancestral allele T(+) for the *P2RX7* associated with the development of ocular disease. Therefore, it is reasonable to speculate that the ancestral allele (that do not carry the rs1718119 SNP and do not contain the Ala-348 to Thr polymorphism) would help to protect against an exacerbated immune response as a consequence of the gain of function conferred by the rs1718119 SNP, which would lead to an immune response with high levels of pro-inflammatory cytokines, such as IL-1 β and IFN-, and could contribute to retinal tissue damage. Our data provides evidence that the Th-1 specific immune response is centered in the IFN- secretion by PBMC of patients with the different types of retinal/retinochoroidal scar lesions, which also reinforces the supposition that an exacerbated immune response in an environment with prolonged inflammatory mediators

Albuquerque and colleagues described an association between the IFN- +874T/A gene polymorphism with toxoplasmic retinochoroiditis susceptibility [51]. This study is related to the Santa Rita (Barra Mansa) casuistic, as described previously in this chapter. The authors found that AA homozygous individuals showed a 1.62-fold higher risk than other genotypes (AT and TT) for developing toxoplasmic retinochoroiditis [51]. Regarding the IFN- +874T/A gene polymorphism, it was demonstrated that the polymorphism is linked to high and low producer phenotypes [27, 28] and the genotype AA is associated with the phenotype for low IFN- capacity of production in contrast to the AT or TT genotypes that are linked with a phenotype for a middle and high IFN- capacity of production respectively [29]. The T to A polymorphism coincides with a putative NF-kappa B binding site that may have functional consequences for the transcription of the human IFN- gene [27]. In the Santa Rita study, the IFN- production by PMBC of the patients was not reported. However,

#### **5. Immunogenetic studies: Candidate genes on ocular toxoplasmosis**

Studies on genetic association in human toxoplasmosis in the past have provided evidence of associations between human leukocyte antigen (HLA) genes with the susceptibility to toxoplasmic encephalitis in AIDS patients (42) and with the outcome of congenital toxoplasmosis [43]. However, no causal relationships have been proven so far. We have conducted genetic association studies of candidate genes which potentially influence the profile of the inflammatory response against *T. gondii* (17, 18, 37) in patients with ocular toxoplasmosis by means of single nucleotide polymorphisms (SNPs) analysis. Significant associations between genetic polymorphisms and ocular disease in family-based studies have been found for Toll-like receptor-9, (*TLR-9*) [17], P2X7 purinoceptor 7 (*P2RX7*) [18] and nucleotide-binding oligomerization domain-containing protein 2 (*NOD2*) [37] genes. These studies are currently being expanded to be replicated into population n-based investigational projects. All of these genes are related to innate immunity and have been described in processes of inflammasome assembly, which plays an important role in processing IL-1 beta and other IL-1 beta cytokine family member (IL-18, IL-33) precursors in active cytokines, promoting a pro-inflammatory response [44].The SNPs rs352140 at *TLR-9* and rs3135499 at *NOD2* were statistically significantly associated with the manifestation of ocular toxoplasmosis [17, 37] while the absence of the SNP rs1718119 at *P2RX7* was strongly associated with protection to ocular disease manifestation [18] as we shall see in detail.

The product of the *P2RX7* belongs to the family of purinoceptors for ATP, 595 amino acids in length and highly polymorphic. The relative amount of P2X7 function varies between human individuals because of the numerous single nucleotide polymorphisms; combinations of these polymorphisms give rise to various haplotypes that can modify P2X7 function and result in either loss or gain of function [45]. Splice isoforms that can alter receptor expression and function and modify the signaling properties downstream of receptor have also been described [45].

The receptor functions as a ligand-gated ion channel and is responsible for ATP-dependent lysis of macrophages by means of the formation of membrane pores that are permeable to large molecules. The receptor P2X7 functions as a pro-inflammatory receptor in cells of the monocyte/macrophage lineage and is activated by extracellular ATP released from a variety of cellular sources including platelets and damaged cells [46] Its expression is up-regulated by IFN-γ and can lead directly to the killing of intracellular pathogens including *T. gondii* [46, 47]. P2X7 stimulates inflammasome activation and secretion of IL-1β [48].

and *T. gondii* infections.

receptor have also been described [45].

valid model for studying parasites that evoke polar Th-1 and Th-2 in mice. *H. polygyrus* is a gastrointestinal worm, a natural parasite to mice, which evokes a polarized Th2 response in the host and blocks the type 1 immune response [39, 40]. It has been shown that a previous infection with the helminth can inhibit the development of CD8 T-cell immune response against *T. gondii*, thus compromising long-term protection against a protozoan parasite [41], which illustrates the adequacy of the model for studying the interaction between helminthic

**5. Immunogenetic studies: Candidate genes on ocular toxoplasmosis** 

associated with protection to ocular disease manifestation [18] as we shall see in detail.

The product of the *P2RX7* belongs to the family of purinoceptors for ATP, 595 amino acids in length and highly polymorphic. The relative amount of P2X7 function varies between human individuals because of the numerous single nucleotide polymorphisms; combinations of these polymorphisms give rise to various haplotypes that can modify P2X7 function and result in either loss or gain of function [45]. Splice isoforms that can alter receptor expression and function and modify the signaling properties downstream of

The receptor functions as a ligand-gated ion channel and is responsible for ATP-dependent lysis of macrophages by means of the formation of membrane pores that are permeable to large molecules. The receptor P2X7 functions as a pro-inflammatory receptor in cells of the monocyte/macrophage lineage and is activated by extracellular ATP released from a variety of cellular sources including platelets and damaged cells [46] Its expression is up-regulated by IFN-γ and can lead directly to the killing of intracellular pathogens including *T. gondii*

[46, 47]. P2X7 stimulates inflammasome activation and secretion of IL-1β [48].

Studies on genetic association in human toxoplasmosis in the past have provided evidence of associations between human leukocyte antigen (HLA) genes with the susceptibility to toxoplasmic encephalitis in AIDS patients (42) and with the outcome of congenital toxoplasmosis [43]. However, no causal relationships have been proven so far. We have conducted genetic association studies of candidate genes which potentially influence the profile of the inflammatory response against *T. gondii* (17, 18, 37) in patients with ocular toxoplasmosis by means of single nucleotide polymorphisms (SNPs) analysis. Significant associations between genetic polymorphisms and ocular disease in family-based studies have been found for Toll-like receptor-9, (*TLR-9*) [17], P2X7 purinoceptor 7 (*P2RX7*) [18] and nucleotide-binding oligomerization domain-containing protein 2 (*NOD2*) [37] genes. These studies are currently being expanded to be replicated into population n-based investigational projects. All of these genes are related to innate immunity and have been described in processes of inflammasome assembly, which plays an important role in processing IL-1 beta and other IL-1 beta cytokine family member (IL-18, IL-33) precursors in active cytokines, promoting a pro-inflammatory response [44].The SNPs rs352140 at *TLR-9* and rs3135499 at *NOD2* were statistically significantly associated with the manifestation of ocular toxoplasmosis [17, 37] while the absence of the SNP rs1718119 at *P2RX7* was strongly Polymorphisms at the *P2RX7* have been investigated in a cooperative immunogenetic study involving patients from United State and Campos dos Goytacazes in Brazil. The studies comprised 149 child in 149 child/parent trios from North America and 60 families with 68 affected with postnatal ocular toxoplasmosis offspring in Brazil [18]. For the United states casuistic, an association between the derived C(+)G(−) allele and a second synonymous variant rs1621388 in linkage disequilibrium with it; and clinical signs of disease *per se.* were observed . Analysis of clinical sub-groups showed associations with retinal disease and brain calcifications (OR=3.0 to 4.25; 0.004<P<0.009). The association with toxoplasmic retinochoroiditis was replicated in a family-based study from Campos dos Goytacazes (60 families; 68 affected offspring), where the ancestral T(+) allele (f= 0.296) at SNP rs1718119 which contains the Ala-348 to Thr polymorphism was strongly protective (OR= 0.27; 95% CI: 0.09–0.80) [18]. This last association at SNP rs1718119 was recently replicated in a casecontrol study with 361 non-related individuals. The study confirmed the protective association of the T(+) allele (f= 0.296) (OR =0,3; 95% CI: 0,15-0,59 chi-square value = 13,53 *p* corrected for multiple comparisons = 0.0132) for patients with type B scar lesions [49]

The SNPs at the *P2RX7* may be associated with a gain or loss of function of the receptor. Data from Stokes and colleagues [50] showed the gain of function for P2X7 haplotypes carrying rs1718119 SNP, which matches our recent case-control data that confirmed a strong protection due to the ancestral allele T(+) for the *P2RX7* associated with the development of ocular disease. Therefore, it is reasonable to speculate that the ancestral allele (that do not carry the rs1718119 SNP and do not contain the Ala-348 to Thr polymorphism) would help to protect against an exacerbated immune response as a consequence of the gain of function conferred by the rs1718119 SNP, which would lead to an immune response with high levels of pro-inflammatory cytokines, such as IL-1 β and IFN-, and could contribute to retinal tissue damage. Our data provides evidence that the Th-1 specific immune response is centered in the IFN- secretion by PBMC of patients with the different types of retinal/retinochoroidal scar lesions, which also reinforces the supposition that an exacerbated immune response in an environment with prolonged inflammatory mediators can contribute to retinal/retinochoroidal damage.

Albuquerque and colleagues described an association between the IFN- +874T/A gene polymorphism with toxoplasmic retinochoroiditis susceptibility [51]. This study is related to the Santa Rita (Barra Mansa) casuistic, as described previously in this chapter. The authors found that AA homozygous individuals showed a 1.62-fold higher risk than other genotypes (AT and TT) for developing toxoplasmic retinochoroiditis [51]. Regarding the IFN- +874T/A gene polymorphism, it was demonstrated that the polymorphism is linked to high and low producer phenotypes [27, 28] and the genotype AA is associated with the phenotype for low IFN- capacity of production in contrast to the AT or TT genotypes that are linked with a phenotype for a middle and high IFN- capacity of production respectively [29]. The T to A polymorphism coincides with a putative NF-kappa B binding site that may have functional consequences for the transcription of the human IFN- gene [27]. In the Santa Rita study, the IFN- production by PMBC of the patients was not reported. However, considering that this cytokine is a very important to parasite control replication, the patients presenting the AA genotype for the IFN- +874 T/A polymorphism likely have a similar profile of specific cellular immune response with that observed for the group that presented lesions of types B and BC, as shown in Figure 3, and multiple type scar lesions, as shown in Figure 4. Namely, they would tend to produce moderate to low levels of IFN- and IL-13 and low levels of CXCL 10, which most likely causes an immune response that is not sufficient to efficiently prevent/control parasite proliferation; as a consequence, retinal/retinochoroidal tissues damage occurs. It is important to note that to determine the frequency of the genotype AA among individuals grouped by the type of scar lesions, in association with phenotypic parameters of the immune response, like cytokines and chemokines, would be of value to improve our understanding of the possible pathological mechanisms that occur in the different types of scar lesions in ocular toxoplasmosis.

Immunological and Immunogenetic Parameters on the Diversity of Ocular Toxoplasmosis: Evidence to Support Morphological Criteria to Classify Retinal/Retinochoroidal Scar Lesions in Epidemiologic Surveys 167

demonstrated to confer a gain of function to the receptor P2X7, imbuing it with a more vigorous pro-inflammatory response. Therefore, the presence of the ancestral allele would protect the eye tissues from harmful exacerbations of immune responses, which corroborates our previous interpretations from PBMC experiments, as described above. Furthermore, the similarity of immunologic parameters between the mouse model and human infection, such as that observed for IFN- and CXCL10, motivates the development of innovative protocols using PBMC to access the gene response at the patient level. This approach is both feasible and suitable. For instance, in a given patient, it is possible to study PBMC to access the expression of the P2X7 protein, the product of the same *P2RX7* that is expressed in retinal epithelial cells. Concerning the relevance of this approach, it is worth mentioning that primary cultures of human retinal epithelial cells that express P2X7 protein [55] are responsive to IFN-γ stimulation leading to *T. gondi*i elimination in a pathway independent of nitric oxide (NO) [36]. The interpretations of our data on PBMC *in vitro* stimulation with *T. gondii* antigens together with the candidate genes genotyping relevant to the innate immune response, targeting the parasite, contribute to a better understanding of the immunological pathogenesis of infectious diseases that has been recently achieved. However, many issues remain to be addressed before the clinical exploitation of these findings can be realized. The function of these elements and their interplay with one another and with other components in the immune system are complex, and it is crucial to determine the balance between their

Finally, the immune responses of patients from population-based studies who exhibit diverse scar lesions that are likely caused by *T. gondii* infection illustrate the multifactorial nature of ocular toxoplasmosis. Beyond host genetics and interactions with other pathogens that we have mentioned in this chapter, there are many other factors, such as environmental questions, the age at which individuals are infected, the parasitic load in the host, and the parasite genetic background that are equally important for disease manifestation. Therefore, all of these aspects, in addition to those not mentioned here or unknown, that characterize the multifactorial nature of ocular toxoplasmosis cannot be considered separately, and their interaction in and with the host will be expressed by one single feature that represents the profile of the immune response mounted by the host. The immune response will be subject to influences by every factor and

, Alba L.P. Rangel, Marcela S.B. Boechat,

will ultimately impact the different clinical presentation of ocular toxoplasmosis.

*Laboratório de Biologia do Reconhecer (LBR) Centro de Biociências e Biotecnologia (CBB) Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes,* 

Bianca M. Mangiavacchi\*\*, Livia M. Martins\*\*, Francielle B. Ferraz\*\*,

beneficial and pathological roles in ocular toxoplasmosis.

**Author details** 

 Corresponding Author \*\* Equal Contribution

*RJ, Brazil* 

 \*

Lílian M.G. Bahia-Oliveira\*

Maycon B. Almeida and Flavia P. Vieira

Other reports in the literature have described genetic association studies of cytokines with toxoplasmic ocular diseases in Brazil. However, they describe small casuistic related to patients from ophthalmologic reference centers and do not find significant associations [52- 54], although one suggests the association of polymorphism at IL-1 alfa gene and the recurrence of ocular toxoplasmosis [54].

### **6. Conclusion**

We have presented data related to a decade of research on a hyperendemic area to *T. gondii* infection, considering aspects linked to the clinical presentation of ocular disease in a population exposed to high risk of waterborne toxoplasmosis, the profile of *in vitro* specific immune response in the function of the disease's clinical presentation, and genetic association studies with candidate genes in ocular toxoplasmosis.

It is important to consider the conclusions and advances that can be derived based on the study of PBMC from patients who exhibit different clinical presentations of ocular toxoplasmosis, stimulated *in vitro* with parasite antigens. One can argue that the study of the specific responses of PBMC *in vitro* does not reflect the eye's immune-privileged environment, for instance. However, the study of PBMC represents is a non-invasive approach that is adequate for population-based studies such that in the genomic medicine era, it will improve our understanding of relevant gene response, i.e., their activation and regulation in inflammatory process within the eye. The associations between phenotype and genotype data from cohorts accelerate our understanding of the molecular mechanisms involved in the disease's pathology. Our data on P2X7 genotyping genes together with the PBMC immune profile illustrate this aspect. Namely, our initial hypothesis on an exacerbation of the inflammatory response with high levels of pro-inflammatory cytokines, especially IFN secretion, concomitant with relatively low levels of anti-inflammatory cytokines like IL-13 secretion in response to parasite antigens, which was identified by *in vitro* PBMC stimulation experiments and could contribute to the development of ocular disease, is reinforced by the findings on *P2RX7* genotypingat the SNP rs1718119. This data showed that the ancestral allele is highly protective for the development of ocular disease. The SNP rs1718119 has been demonstrated to confer a gain of function to the receptor P2X7, imbuing it with a more vigorous pro-inflammatory response. Therefore, the presence of the ancestral allele would protect the eye tissues from harmful exacerbations of immune responses, which corroborates our previous interpretations from PBMC experiments, as described above. Furthermore, the similarity of immunologic parameters between the mouse model and human infection, such as that observed for IFN- and CXCL10, motivates the development of innovative protocols using PBMC to access the gene response at the patient level. This approach is both feasible and suitable. For instance, in a given patient, it is possible to study PBMC to access the expression of the P2X7 protein, the product of the same *P2RX7* that is expressed in retinal epithelial cells. Concerning the relevance of this approach, it is worth mentioning that primary cultures of human retinal epithelial cells that express P2X7 protein [55] are responsive to IFN-γ stimulation leading to *T. gondi*i elimination in a pathway independent of nitric oxide (NO) [36].

The interpretations of our data on PBMC *in vitro* stimulation with *T. gondii* antigens together with the candidate genes genotyping relevant to the innate immune response, targeting the parasite, contribute to a better understanding of the immunological pathogenesis of infectious diseases that has been recently achieved. However, many issues remain to be addressed before the clinical exploitation of these findings can be realized. The function of these elements and their interplay with one another and with other components in the immune system are complex, and it is crucial to determine the balance between their beneficial and pathological roles in ocular toxoplasmosis.

Finally, the immune responses of patients from population-based studies who exhibit diverse scar lesions that are likely caused by *T. gondii* infection illustrate the multifactorial nature of ocular toxoplasmosis. Beyond host genetics and interactions with other pathogens that we have mentioned in this chapter, there are many other factors, such as environmental questions, the age at which individuals are infected, the parasitic load in the host, and the parasite genetic background that are equally important for disease manifestation. Therefore, all of these aspects, in addition to those not mentioned here or unknown, that characterize the multifactorial nature of ocular toxoplasmosis cannot be considered separately, and their interaction in and with the host will be expressed by one single feature that represents the profile of the immune response mounted by the host. The immune response will be subject to influences by every factor and will ultimately impact the different clinical presentation of ocular toxoplasmosis.
