Congenital Toxoplasmosis: Candidate Host Immune Genes Relevant for Vertical Transmission and Pathogenesis

REVIEW Congenital toxoplasmosis: candidate host immune genes relevant for vertical transmission and pathogenesis

LB Ortiz-Alegrıá1, H Caballero-Ortega1, I Can˜edo-Solares1, CP Rico-Torres1, A Sahagu´ n-Ruiz2, ME Medina-Escutia3 and D Correa1 1Laboratorio de Inmunologıá Experimental, Subdireccioń de Medicina Experimental, Instituto Nacional de Pediatrıá, SSA, Me´xico DF, Mexico; 2Departamento de Microbiologıá e Inmunologıá, Facultad de Medicina Veterinaria y Zootecnia, UNAM, Me´xico DF, Mexico and 3Laboratorio de Anticuerpos Monoclonales, Instituto de Diagno´stico y Referencia Epidemiolo´gicos, SSA, Me´xico DF, Mexico

Toxoplasma gondii infects a variety of vertebrate hosts, including humans. Transplacental passage of the parasite leads to congenital toxoplasmosis. A primary infection during the first weeks of gestation causes vertical transmission at low rate, although it causes major damage to the embryo. Transmission frequency increases to near 80% by the end of pregnancy, but the proportion of ill newborns is low. For transmission and pathogenesis, the parasite genetics is certainly important. Several host innate and adaptative immune response genes are induced during infection in adults, which control the rapidly replicating tachyzoite. The T helper 1 (Th1) response is protective, although it has to be modulated to avoid inflammatory damage. Paradoxical observations on this response pattern in congenital toxoplasmosis have been reported, as it may be protective or deleterious, inducing sterile abortion or favoring parasite transplacental passage. Regarding pregnancy, an early Th1 microenvironment is important for control of infectious diseases and successful implantation, although it has to be regulated to support trophoblast survival. Polymorphism of genes involved in these parallel phenomena, such as Toll-like receptors (TLRs), adhesins, cytokines, chemokines or their receptors, immunoglobulins or Fc receptors (FcRs), might be important in susceptibility for T. gondii vertical transmission, abortion or fetal pathology. In this study some examples are presented and discussed. Genes and Immunity (2010) 11, 363–373; doi:10.1038/gene.2010.21; published online 6 May 2010

Keywords: congenital toxoplasmosis; host genetics; immune response; polymorphisms; Toxoplasma gondii

Introduction likely to influence outcome. Recent investigations have shown surprising phenomena; that is, molecules and The protozoan parasite Toxoplasma gondii, an obligate cells that protect the mother might favor vertical intracellular eukaryotic pathogen of the phylum Api- transmission.6,7 Few direct data are available, but indirect complexa, may cause toxoplasmosis in many warm- evidence points to several candidate polymorphic host blooded animals, including humans.1 Transplacental immune response genes that may influence fetal infec- passage of the parasite causes congenital toxoplasmosis. tion or clinical outcome of the product. Transmission frequency and severity of disease vary with gestation time: during the first weeks, vertical Immune response in acquired toxoplasmosis transmission is of low rate, although if it occurs, it causes The key immune system molecules and cells related to major damage to the embryo. The transmission fre- control acquired (noncongenital) toxoplasmosis have quency increases to near 80% by the end of pregnancy, been studied extensively. It can be summarily said that 2 but the proportion of ill newborns is low. The changes in professional phagocytes and natural killer (NK) cells are endocrine phenomena occurring during pregnancy, as main effectors during the innate phase. Human Toll-like well as the size and maturity of the placenta and of the receptors (TLRs) of importance are TLR2 and TLR4. embryonic/fetal immune response certainly affect the Their ligands induce the production of inflammatory ability to be protected from invasion or to fight chemokines and cytokines (that is, CCL3, CCL5, inter- 3–5 infection. The size of the inoculum is also relevant leukin (IL)-6, IL-8 and IL-12) that mediate recruitment of for congenital infection risk and disease severity. Besides, professional phagocytes and dendritic cells.8–10 Crucial the genetic background of the mother and the product is adaptative responses include CD8 þ cytotoxicity of infected cells, antibody-mediated phagocytosis and NK cell antibody-mediated cytotoxicity (NK-ADCC). Correspondence: Dr D Correa, Lab. Inmunologıá Experimental, A T helper 1 (Th1) profile (that is, IL-12 and interferon-g Instituto Nacional de Pediatrıá, SSA, Av. Insurgentes Sur 3700-C, (IFN-g)) is essential to induce these mechanisms.11 Th17 Torre de Investigacioń, Mexico DF, 04530, Mexico. E-mail: [email protected] cells are also involved in acute polymorphonuclear- Received 10 November 2009; revised and accepted 16 March 2010; mediated resistance to Toxoplasma.12 Some chemokines published online 6 May 2010 and their receptors have a determinant role for NK and Congenital toxoplasmosis LB Ortiz-Alegrıá et al 364 dendritic cell recruitment; in addition, polarization to promote fetal growth and to inhibit allorejection by a Th1 profile depends on CCR5 stimulation by T. gondii maternal immune response. By the end of pregnancy, a cyclophilin 18, CCL3, CCL4 or CCL5.13,14 Other CCRs, Th1 inflammatory response arises again, promoting rup- such as CCR1, CCR2 and CXCR2, have important roles ture of membranes, placental detachment and partum.4,36 in neutrophil proliferation and monocyte recruitment, which are essential for parasite control.15–18 An early Th2 Candidate immune genes with a role in T. gondii vertical phenotype (driven by IL-4) is associated with bad transmission or fetal pathology outcome.19,20 HLA. The major histocompatibility complex (MHC) Antibodies and their Fc receptors (FcRs) have im- antigens have an important role in acquired toxoplasmosis portant roles as well.21,22 Antibodies of the immunoglo- for both CD8 þ lymphocyte-mediated cytolysis and for bulin (Ig)A, IgM and IgG1 (equivalent to murine IgG2a) CD4 þ -mediated regulation of the immune response. classes protect through neutralization, complement acti- Many studies using the murine model have described the vation, opsonization of phagocytes or NK-ADCC. IgG2 role of these antigens in the control of parasite replication and IgG4 are poor complement fixative subclasses and of or generation of inflammatory disease.11 Thus, MHC low affinity for FcRs, and thus they are not expected to antigens are thought to have a major role in fetus suscep- relate to protection. Similarly, IgE antibodies are related tibility to develop clinical problems. In this regard, Mack to lymphadenopathy in the acute phase.22 et al.37 showed that among congenitally infected Caucasian Once the parasite encysts, the Th1/Th17 response- children, the DQ3 allele frequency was significantly higher driven inflammatory reactions are downregulated, avoid- in infants with hydrocephalus than in those without it. In ing inflammatory damage. This is mediated by IL-10, transgenic chimerical mice they also showed that the transforming growth factor-b (TGF-b) and IL-27.11,23,24 human DQ3 allele protected less than the DQ1 against brain necrosis.37 Immune response and pregnancy Immunohistochemically, HLA-G and a small amount Thereissomeparallelismbetweentheresponsethat of HLA-C are the only molecules that can be detected in controls T. gondii infection and that related to pregnancy extravillous chorionic cytotrophoblasts.25 Its low poly- success. During implantation, a Th1-type inflammatory morphism ensures that paternal HLA-G expressed in the environment is essential for invasion of the endometrium trophoblast does not induce a maternal alloresponse by by trophoblasts; it is also necessary during the first month the class I MHC-restricted CD8 þ cells. Conversely, of pregnancy to favor spiral arteries growth and protection activation of NK cells depends on the lack of MHC from infection.25,26 Implantation is induced by IFN-g, expression in the target cells, but extravillous cytotro- which activates NK cells and regulates colony stimulation phoblast HLA-G expression induces IL-10 and other factors, promoting trophoblast growth. Chemokine and cytokines, which build a favorable milieu for pregnancy chemokine receptor signaling processes mediate leukocyte maintenance.38 Thus, the trophoblast resists NK cell and recruitment and controlled trophoblast invasion and thus CD8 þ lysis by disturbed antigen recognition through implantation and placentation.27 CCR1 is present on HLA-G, although low diversity peptides are presented invading human trophoblasts and promotes their migra- by HLA-G to cytotoxic T lymphocytes.38 Nothing is tion. Its ligand, CCL14, is expressed in endometrial known about T. gondii antigen presentation through vasculature, as well as in epithelial and decidual cells HLA-G, but it would induce limited CD8 þ lysis of during early pregnancy. CCR2 and CCL2 are coexpressed infected cells and Th1-type inflammation during early by human first-trimester decidual tissue and the latter is pregnancy, which would partially favor local parasite upregulated by pregnancy-associated hormones, such as control and implantation success. estrogen, progesterone and human chorionic gonadotrophin.28 CCL5 promotes a Th1 profile and an adequate pro- Cytokines, chemokines, adhesion molecules and receptors implantatory microenvironment, influencing trophoblast As IFN-g and related cytokines and chemokines are key cell survival and balance of maternal T regulatory/T molecules for protection against acquired toxoplasmosis effector lymphocytes in favor of maternal tolerance.29 and successful implantation of the embryo, a positive CXCL12/CXCR4 crosslinking stimulates antiapoptotic relation between inflammation degree and protection pathways in cultured trophoblasts, suggesting that they from T. gondii vertical transmission would be ex- are important in trophoblast survival.30 CX3CR1 is present pected.36,39,40 In fact, the inflammatory cells recruited by on invading human trophoblasts and promotes their Th1 profile-associated chemokines and cytokines pro- migration by action of its ligand CX3CL1, which is tects the embryo from infections.4,26 Nevertheless, recent expressed by endometrial vasculature, epithelial and data on the effect of IFN-g on congenital toxoplasmosis decidual cells during early pregnancy.31 This early in mouse models indicate that extremely high levels may inflammation is downregulated, and hence the invading induce resorption of the embryo without actual infection trophoblasts do not overgrow and destroy the supporting of the placenta.41 This implies that sterile abortion may decidual/endometrial tissues. IL-10 promotes implanta- be induced indirectly by T. gondii in mothers with strong tion and development of the fetus. It also influences Th1 or low IL-10/TGF-b/IL-27 responses. Polymorph- human leukocyte antigen (HLA) class I expression at the isms related to strong Th1 inflammatory response would feto–maternal barrier, which protects the fetus from hypothetically cause this clinical situation, similar to the rejection. TGF-b is the major inhibitor of cytotrophoblast IFN-g gene promoter mutation T-179G, which causes a outgrowth, fusion and syncytialization. It inhibits the 6- to 13-fold higher expression by T cells (Table 1).42,43 production of human chorionic gonadotrophin, human Direct invasion of placental cells by T. gondii is placental lactogen, progesterone and oestradiol.32–35 regulated by immune response molecules. Barbosa During most of the gestation period, a delicate balance of et al.44 showed that BeWo line trophoblasts are suscep- the Th1/Th2/regulatory T cell cytokines prevails to tible to invasion and replication, especially if they are

Genes and Immunity Table 1 Human immune response genes: hypothetical effect of some polymorphisms on congenital toxoplasmosis

Gene product Producer cells in the mother Producer cells/tissue Number of Polymorphism Polymorphism effect Hypothetical effect on vertical transmission or fetal References (NCBI gene IDa ) in the placenta reported example clinical outcome SNPsa

CCL5, RANTES Stim-TCs First-trimester villi 115 À28 C/G or G/G Increased transcription Strong inflammatory reaction; good balance and 69 (6352) (rs1800825) successful control of invasion or enhanced inflammation and lower survival of trophoblasts: probable sterile abortion? À403A (rs2107538) Low RANTES production 70 CCR2 (729230) Bas, DCs, Mfs, NKs, First-trimester 126 V64I (rs1799864) Expression of nonfunctional receptor 65 Stim-TCs decidual tissue CCR5 (1234) DCs, Mon, NKs, Stim-TCs Trophoblast 134 D32 (rs56030631) Truncated protein. Reduced expression in Weak Th1/proimplantatory environment; low 65–67 heterozygous control of parasite replication; larger chances of vertical transmission. T. gondii infection may 73–75,77 TLR-2 (7097) BCs, EDs, Mfs, Mon, HbCs, trophoblast, 193 T-16934A Diminished synthesis of TNF-a and IFN-g. counteract polymorphism effect due to induction Neu, NKs syncytiotrophoblast, (rs4696480) Potentially leads to aberrant innate immune of Th1 response. Polymorphism in either the uNKs responses, which may contribute to early mother or the embryo/fetus could be important preterm birth TLR-4 (7099) DCs, EDs, Mfs, Mon, Neu 225 Asp299Gly Low levels of IL-6, IL-10 and Th1 cytokines 73,74–77 (rs4986790)/ Thr399Ile IFN-g (3458) NKs, TCs uNKs 8 T-179G 6- to 13-fold induction of IFN-g in response Strong inflammatory reaction; good balance and 42, 43 to TNF-a successful control of invasion or enhanced inflammation and diminished survival of trophoblasts: sterile abortion if not modulated TNF-a (7124) Mfs Decidual Mfs 139 G-308A (TNFa1/ Elevated TNF-a levels 60 TNFa2) 35,45,46 TGF-b (7040) Mfs, TCs Cytotrophoblast, 319 Pro25Arg Homozygous Arg associated with higher Negatively associated with local inflammation Ortiz-Alegrı LB toxoplasmosis Congenital syncytiotrophoblast (rs1800471) TGF-b1 level than a heterozygous and implantation process. Polymorphisms may favor parasite invasion and replication in syncytiotrophoblast IL-10 (3586) Mfs, TCs Cytotrophoblasts 138 A-1082G, T-819C, Haplotype À1082G, À819C, À592C 32,45–47 and decidua A-592C associated with an increased expression 79

IL-4 (3565) MCs, Mfs, TCs Cytotrophoblast, 211 C-590T (rs2243250) Increased expression Negatively associated with Th1 inflammation. ´ FECs Polymorphism might favor local damage. In a infected fetuses it can lead to non-protective al et cellular and humoral (IgE or IgG2) response; thus, associated with clinical problems, depending on time of gestation IL-6 (3569) Mfs Trophoblast, 158 C -174G Increased IL-6 production in homozygous 80,81 syncytiotrophoblast, (rs13447445) maternal decidua ICAM-1 (3383) BCs, EDs, Mon, TCs Syncytiotrophoblast, 238 ICAM-1 Kilifi Reduced avidity for ligand LFA-1 Lower adhesion of infected monocytes: lower 56,57 cytotrophoblast, (rs5491) vertical transmission probability FECs MIF (4282) EDs, Mfs First-trimester 251 Haplotype G-173C Increase in expression, higher levels secreted High-expression alleles would augment 58 trophoblast and (rs755622)/CATT- expression on syncytiotrophoblast and increase decidua repeat at À794 ICAM-1-related enhanced vertical transmission

Abbreviations: Bas, basophil; BC, B lymphocyte; DC, dendritic cell; ED, endothelial cell; FEC, fetal endothelial cell; HbC, hofbauer cell; ICAM-1, intercellular adhesion molecule 1; IFN-g, interferon-g; IL, interleukin; LEC, lens epithelial cell; LFA-1, lymphocyte function-associated antigen-1; MC, mast cell; Mf, macrophage; MIF, macrophage migration inhibitory factor; Mon, monocyte; Neu, neutrophil; NK, natural killer cell; SNP, single-nucleotide polymorphism; Stim, stimulated; TC, T lymphocyte; TGF-b, transforming growth factor-b; TLR, Toll-like receptor;

ee n Immunity and Genes TNF-a, tumor necrosis factor-a; uNK, uterine natural killer cell. a http://www.ncbi.nlm.nih.gov/pubmed. 365 Congenital toxoplasmosis LB Ortiz-Alegrı´a et al 366 treated with IFN-g, IL-10 and TGF-b1. When endogenous T. gondii acute infection. They affect susceptibility or IL-10 or TGF-b were blocked, the cells controlled parasite resistance to several infectious diseases.45,46,61–64 In addi- infection only in the presence of IFN-g. If susceptibility of tion, chemokine receptor signaling processes are impor- BeWo cells to T. gondii is because of immunomodu- tant for leukocyte recruitment and controlled trophoblast lation mechanisms, the role of trophoblast cells in invasion and thus for implantation and placentation.25,26 maintaining a placental microenvironment favorable to CCR2 and CCR5 are chemokine receptors that have been pregnancy may facilitate local infection, especially for assigned a function in toxoplasmosis and, independently those mothers with high expression alleles of the in pregnancy, they protect from uncontrolled T. gondii modulating cytokines (that is, TGF-b 25Pro and IL-10 replication in adults and favor trophoblast survival or haplotype À1082G/À819C/À592C; Table 1).32,35,45–47 It is invasion (related to a Th1 response), partially explaining important to emphasize that BeWo cells are not models the first-trimester resistance against vertical transmis- for the syncytiotrophoblast, the main mother/conceptus sion.11,13,14,25–27 The 64I allele of CCR2 and the D32 interface. Thus, these results would be related to local deletion of CCR5 negatively influence expression of the invasion of the placenta, but not necessarily to passage products, which have shown implications in noninfec- across the mother/embryo barriers. tious diseases and, of special interest, human immuno- A paradoxical phenomenon has been observed in deficiency virus congenital transmission.65–67 Similarly, animal and in vitro models; that is, IFN-g may support CCL5 (RANTES) production in response to Brucella vertical transmission at the time it protects the mother abortus correlated with IFN-g induction and contributed from parasite-induced damage.6,48 Treatment of human to abortion in mice.68 Two polymorphisms of CCL5 in placenta villous explants with soluble antigen from either the mother or the product would have opposite T. gondii plus IFN-g significantly induces parasitized effects on congenital toxoplasmosis occurrence or white blood cell adhesion to placental villous cells, pathology: the À28 C/G or G/G would be protective augmenting in this way vertical transmission probabil- against infection, but without proper control it could ity.49,50 Pretreatment of syncytiotrophoblast with the relate to sterile abortion.69 On the contrary, carriers of the inflammatory cytokines IFN-g, tumor necrosis factor-a À403A allele would have low CCL5 levels and thus (TNF-a) and IL-1a increases the number of monocytes people with this allele would have higher risk of vertical bound to the surface by upregulation of intercellular transmission.70 adhesion molecule 1 (ICAM-1).49,50 An interesting find- The recognition of T. gondii pathogen-associated ing indicates that the parasite may directly adhere to the molecular patterns is mediated by TLR2, TLR4 whose syncytiotrophoblast through the adhesin called micro- signaling cascades are implicated in eliciting IL-12 and neme 2 (MIC2), which binds to human ICAM-1.51 TNF-a. TLR2 specifically recognizes glycosylphosphati- Another molecule implicated in this phenomenon is dylinositol of the tachyzoites.9,11,71,72 TLR2 and TLR4 are macrophage migration inhibitory factor (MIF), which is expressed on the surface of the syncytiotrophoblast produced by first-trimester placental trophoblasts, de- that recognizes microbes and produces TNF-a, IL-6 and cidua and cytotrophoblasts, and is implicated in angio- IL-8.73,74 Intrauterine infections may result in either genesis, cell proliferation, and thus in implantation and trophoblast apoptosis or cytokine production that signal early embryonic development.52 Intravillous cytotropho- immune cells and promote trophoblast survival and blasts produce MIF in response to T. gondii soluble destruction of infectious agents. A polymorphism in the extract; and then, ICAM-1 is induced in the syncytio- TLR2 gene 16934A is linked to diminished synthesis of trophoblast maternal face.53–55 Hypothetically, the ma- TNF-a and IFN-g. This potentially leads to aberrant ternal À179T low-expression allele of IFN-g, as well as innate immune responses, which may contribute to early the fetal Kilifi allele of ICAM-1 or the low-expression preterm birth.75 Individuals possessing the TLR4 haplotype of MIF, would be compatible with resistance co-segregating polymorphisms, Asp299Gly and against vertical transmission (Table 1).43,56–58 Thr399Ile, are hyporesponsive to lipopolysacharide and TNF-a is a key cytokine in acquired toxoplasmosis are more susceptible to Gram-negative bacterial infec- control. It is mainly produced by macrophages and tions.73,76,77 Those fetuses carrying the low-expression mediates phagocytosis enhancing and damage to in- alleles of these receptors would hypothetically be more fected cells.59 At the mother/embryo interface, it is susceptible to die either by uncontrolled parasite expressed by maternal decidual macrophages, in which replication/invasion or impaired trophoblast growth. it regulates fetal trophoblast growth and invasion. Regulation of most Th1 effects is mainly mediated by Abnormal levels are associated with amniotic infections, IL-10 and TGF-b. In vitro, the haplotype À1082G/ recurrent spontaneous abortions, preeclampsia, preterm À819C/À592C of the IL-10 gene is associated with an labor and endometriosis. Several single-nucleotide poly- increase in the protein amount. Among reported single- morphisms are known for TNF-a. Polymorphism –308A nucleotide polymorphisms of TGF-b gene, the Arg25 is has been related to TNF-a overexpression (Table 1).60 also related with an increased expression.35,45,46 High- After a maternal infection it could have two effects: high expression polymorphisms of either TGF-b or IL-10 levels would overinduce implantation, thereby inducing would protect the implanting embryo against sterile sterile embryonic loss, but in low-expression allele abortion or IFN-g/ICAM-1-facilitated vertical transmis- carrier mothers, it could be ineffective to control sion, but would be negative for parasite-induced infection, allowing parasite vertical transmission or pathogenesis control in the conceptus already infected destruction of the early placenta. by T. gondii. The classical Th2 interleukin, IL-4, has been Several polymorphisms have been described in genes shown to be nonprotective in T. gondii-acquired infection of chemokines, TLRs or regulatory cytokines, such as in both humans and animals.19,20 It stimulates cellular IL-4, TGF-b, IL-10 or their receptors, that might mechanisms not protective against intracellular para- cause differences among mother/newborn pairs during sites, and even induces undesirable effects, similar to IgE

Genes and Immunity Congenital toxoplasmosis LB Ortiz-Alegrı´a et al 367 antibodies or Th2 inflammation. It is produced by for infectious diseases.89–91 In both cases, phagocytosis is cytotrophoblasts, decidual macrophages and both ma- compromised. The FcgRIIIa presents a triallelic change: ternal and fetal endothelial cells (Table 1).78 Polymorph- 48Arg and 48His present stronger binding for human ism À590TT of this gene is related with asthma and atopy IgG1, IgG3 and IgG4, than 48Leu.92 The FcgRIIIb has a because of high expression.79 It may contribute to class II biallelic polymorphism, neutrophil antigen 1 (NA1)or2 MHC molecule upregulation on both maternal and fetal (NA2), expressed in polymorphonuclear neutrophils, macrophages, activating antigen-presenting cells in- which are different in four amino acids.93 Homozygous volved in antimicrobial response during pregnancy. NA1 individuals have higher IgG-mediated phagocytosis The proinflammatory IL-6 is produced by tropho- than NA2.91,93 blasts, maternal decidua and chorionic villous of the Fc receptors such as FcgRI, FcgRII, FcgRIII and FcRn placenta. It induces human chorionic gonadotrophin show heterogeneous expression patterns in the placenta liberation by trophoblasts, promotes progesterone and (Table 2). Immunohistochemical analyses reveal the Th2 cytokines production, for example, IL-4, and presence of FcgRI in Hofbauer cells (macrophages) in suppresses Th1 response, and is associated with neonatal stromal tissue within the villous; in peripheral mono- sepsis inflammation.80 The –174G allele correlates with cytes, it mediates endocytosis and degradation of high expression levels of the protein.81 As for other immune complexes, and thus this is probably its function cytokines/chemokines, the effect of polymorphisms in Hofbauer cells.94 FcgRII, which binds IgG complexes, related to increased expression of IL-6 on T. gondii has been found in Hofbauer cells and fetal blood vertical transmission must be considered in terms of the endothelium. Transcripts from FcgRIIa or FcgRIIc are time of pregnancy at the moment of infection, and on its present in placenta throughout pregnancy. FcgRIIb possible dual role; for example, a strong IL-6 response mRNA is detected only after week 20 and its expression would recruit inflammatory cells (phagocytes for exam- by endothelial cells of the human placenta may be of ple) that may fight the parasite, but they would particular immunological importance.94 As this receptor counteract IFN-g action. possesses significant affinity for multivalent or aggre- gated IgG only, it is not yet clear whether endothelial Igs and FcRs polymorphisms cells are responsible for clearing immune complexes Igs and their receptors are crucial against microbial from the fetal circulation or this receptor is mediating agents.21 In addition, the four IgG subclasses are actively transport from the connective stromal tissue to the fetal transported across syncytiotrophoblast and the villous circulation.95 Existing data suggest that FcgRIIIb is fetal endothelial cells, and hence they reach the con- present in first trimester syncytiotrophoblast. The FcgRIII ceptus blood and protect him/her even for several in Hofbauer cells is expected to be FcgRIIIa, which is months after birth.82 Besides, Igs are produced by the detected in macrophages. Both FcgRIIIa and FcgRIIIb human fetus by week 14 of gestation and they may be bind IgGl and IgG3 complexes, but not IgG2 or IgG4.82,94 stimulated by antigens.83,84 Transplacental transport of maternal IgG has classi- Four systems have been described for the constant cally been considered protective for the newborn against heavy and k light chain genes of Igs, Gm, Am, Em and infection, and most of the IgG is transferred during the Km, with 24 allotypes.85,86 Gm and Km allotypes are third trimester (Figure 1).82 Thus, it would be reasonable associated with amino acid substitutions in the constant that vertical transmission was lower by the end of regions—the former influencing IgG1, IgG2 and IgG3 gestation. Nevertheless, it is just the opposite (Figure 2).2 subclass levels—and are inherited in the Mendelian The FcRn is present in the syncytiotrophoblast and fetal manner.87 endothelial cells, and binds IgG of the four subclasses The sizable gene family of human FcRs belongs to the with high affinity.82,94 It mediates IgG transcytosis and large Ig supergene family. Their products show a wide immune complexes during pregnancy.82,88,95 A variable cellular distribution, and through interaction with Igs, number of tandem repeats (VNTR) region within the mediate a variety of leukocyte functions.88 Evidence from promoter, with five different alleles (VNTR1–VNTR5), the rodent toxoplasmosis models and functional corre- has been studied in monocytes; this polymorphism spondence of humans Igs and FcRs to those of mice influences FcRn expression leading to variable cellular suggest that fetal alleles associated with high serum IgG-binding activity. VNTR3 homozygous monocytes levels of Igs, or with strong Ig/FcR binding would be express 1.66-fold more FcRn transcript than VNTR2/ related to better clinical outcome in the fetus, although VNTR3 heterozygous cells, and hence the latter show they could also be related to an exacerbated, that is, diminished IgG binding.96 damaging response.22 Some polymorphisms of the There is a remarkable paradox: pregnant women are human Fc receptors and their hypothetical effects are equally susceptible to T. gondii primo-infection through- listed in Table 2. Eight genes transcribed in different out pregnancy, but the parasite is more frequently mRNAs have been recognized. The FcgRII a, b and c transmitted after the first trimester. Moreover, the genes encode highly homologous proteins regarding the responses that protect the mother from damage favor extracellular parts, but show strong structural diver- vertical transmission: IgG1 antibodies were statistically gence in cytoplasmic domains. Differences in the more frequent among mothers whose newborns pre- subclass specificity between the forms and differential sented clinical signs when compared with those expression on distinct cells have critical consequences for with newborns infected but asymptomatic.7 The hypoth- antibody effector functions (Table 2).21 Two alleles for esis is that FcRn is facilitating rather than protecting from each FcgRIa and FcgRIIa (Arg92Ter and His131Arg, vertical transmission. In this regard, it has been found respectively) have been described. In the first case, that maternal antibodies against cytomegalovirus expression is altered, whereas FcgRIIa-His131 binds may favor fetal infection.97 FcRn could be facilitating IgG2 strongly and is considered a heritable risk factor the ‘opsonized’ tachyzoite traverse the placenta.

Genes and Immunity 368 ee n Immunity and Genes

Table 2 Human Fc receptors: hypothetical effect of some polymorphisms on congenital toxoplasmosis

Gene product (NCBI Binding to Producer cell Producer cells/tissue Number of Polymorphism Polymorphism effect Hypothetical effect on vertical References gene IDa ) in the mother in the placenta reported SNPsa example transmission or fetal clinical outcome IgG1 IgG2 IgG3 IgG4

FcgRIa, CD64 (2209) +++ — ++ (+) Mfs, Mon HbCs (surface) 81 Arg92Ter mRNA stability affected: Clinical problems in the fetus 88 undetectable expression in due to lower phagocytosis capacity phagocytes toxoplasmosis Congenital FcgRIIa, CD32 (2212) +++ +/À +++ + LangCs, Mfs, HbCs (surface) and 343 Arg131His Better IgG2 handling, low 89,90 Mon, Neu, Plat FECs (basolateral) (rs1801274) phagocytosis in Ortiz-Alegrı LB homozygous HH FcgRIIb, CD32 (2213) +++ + ++++ +++ BCs 271 Tyr258Asp Probably diminished Clinical problems due to exacerbated feedback inhibition inflammation in spite of adequate parasite control FcgRIIIa, CD16 ++ +/À + + Mfs, NKs, Stim- HbCs (surface) and 290 Leu 48His/Arg Higher affinity for IgG1 Better control of parasite replication 92 ´

(2214) Mon, TCs syncytiotrophoblast and 3; ADCC augmented by the fetus. a

(apical) Doubtful effect on vertical al et transmission FcgRIIIb, CD16 ++++ + ++++ + Neu, Stim-Eos 108 NA1/NA2 (4 Lower phagocytic Clinical problems in the fetus due to 91,93 (2215) different aa) capacity lower phagocytosis capacity. Doubtful effect on vertical transmission FcgRn (2217) ++++ +++ ++ +++ Mon Syncytiotrophoblast 135 (VNTR1– Low (1) to high (5) Higher probability of transplacental 94,95 (apical) and FECs VNTR5) in expression level passage in VNTR5. Damage to fetus (basolateral) promoter dependent on time of gestation FcaRI, CD89 (2204) IgA DCs, Eos, Mfs, No 223 Ser248Gly High Neu IL-6 Clinical problems in the mother or 98 Mon, Neu (rs16986050) production already-infected fetus, due to lower parasite control by Th2-linked response. No effect on vertical transmission FceRI a chain (2205) IgE Bas, LangCs, No 169 C-315T Significantly larger 99 MCs amount of FceRI

Abbreviations: Bas, basophil; BC, B lymphocyte; DC, dendritic cell; Eos, eosinophil; FEC, fetal endothelial cell; HbC, Hofbauer cell; Ig, immunoglobulin; LangC, Langerhans cell; MC, mast cell; Mf, macrophage; Mon, monocyte; Neu, neutrophil; NK, natural killer cell; Plat, platelet; SNP, single-nucleotide polymorphism; Stim, stimulated; TC, T lymphocyte; Ter, terminal codon. a http://www.ncbi.nlm.nih.gov/pubmed. Congenital toxoplasmosis LB Ortiz-Alegrı´a et al 369 FcγRIIa, FcγRIIc and FcγIIIa Fcγ RIIIb Vertical transmission: Abortion/fetal damage: -Placental invasion: parasite -Invasion/destruction of fetal virulence genes tissues: parasite virulence genes

FcγRn -“Aided” passage through -Sterile abortion: maternal and placenta: fetal genes (stimulated by parasite?) • Mother genes (IFN-γ, -Fetal defense/pathology genes IgG transfer chemokines, TLRs, IgGs)

level FcγRIIb • Fetal genes (ICAM-1, MIF, -Non-immune fetal genes FcγRn) (collagen, ATP transport cassete)

I II III Rate Trimester of pregnancy Rate Figure 1 Schematic representation of Fcg receptor expression levels in placental tissues, maternal IgG transfer to the fetus and rate of T. gondii transmission during pregnancy in humans. IIIIII IIIIII Gestation trimester Figure 2 Diagrammatic summary of polymorphic genes of major relevance for vertical transmission and clinical outcome in Polymorphisms in both the G1m alleles of the mother congenital toxoplasmosis. Seroconversion studies have shown an and the FcRn VNTRs of the fetus may affect the serum increment in vertical transmission rate and a decrease in embryonic/fetal damage during pregnancy. and syncytiotrophoblast membrane levels, respectively, and they would influence the final parasite burden that the developing fetus should confront and thus the clinical outcome. Alternatively, IgG1 antibodies could Non-immune response genes be just markers of the IFN-g profile that induces Many non-immune genes of the host may influence syncytiotrophoblast ICAM-1 expression and enhanced tissue pathology during embryogenesis. Two mole- infection (see above). cules were specifically studied by Jamieson et al102: the IgA receptor I (FcaRI) is expressed in neutrophils, IIB isoform of collagen 2A1, involved in optic nerve monocytes, tissue macrophages, eosinophils and sub- conduction during early development, and the ABCA4 populations of dendritic cells and can activate phagocy- form of the retina-specific ATP-binding cassette trans- tosis and other immune responses. The FcaRI gene porter protein, present in the choroid plexus and at the presents one nonsynonymous single-nucleotide poly- rim of the photoreceptor outer membrane.102 They were morphism in the coding region (248 Ser for Gly) within selected because of their relation to other noninfectious the cytoplasmic tail domain.98 The two alleles have ocular and brain diseases. Studying large European and significantly different FcaRI-mediated intracellular cal- North American cohorts, they found that certain single- cium mobilization and degranulation and cytokine nucleotide polymorphism alleles were associated with production (IL-6 and TNF-a). In addition, IgA may ocular problems in the infected newborns, compared inhibit IL-6 production by neutrophils from human with infected but not affected children. Interestingly, they donors carrying the Ser248 allele. Thus, a-chain alleles also found epigenetic factors affecting disease outcome. of FcaRI affect receptor-mediated signaling and have an important role in immune responses modulation in inflammatory diseases.98 FceRI regulates the concentra- tion of circulating IgE antibodies; among its genetic Concluding remarks variants, the C/T transition at À315 in the promoter of Congenital toxoplasmosis is a very complex phenomen- the a-chain gene is related to increased IgE levels.99 on; the outcome depends of the genetics of three beings: Polymorphisms in these FcaRI and FceRI will probably the mother, the conceptus and the parasite. In addition, affect the ability of the already-infected embryo/fetus to there are two different phenomena remarkably depen- respond properly to T. gondii, but little can be suspected dent on the time of gestation at challenge: the probability about their role in vertical transmission, as they are not of vertical transmission at the moment of maternal present in the syncytiotrophoblast. infection and the pathological effect on the embryo/ Similar to acquired toxoplasmosis, most antibody fetus (Figure 2). Few conclusive results can be gathered classes are expected to protect the fetus against cell from the literature regarding these questions, although invasion by neutralizing, mediating killing of the candidate genes for further study were identified. The parasite by complement fixation or enhancing cellular immune response of the developing baby has a crucial defense mechanisms, especially FcgR-mediated NK- role in tissue destruction avoidance. In this regard, ADCC or macrophage/neutrophil phagocytosis. Only lessons from acquired toxoplasmosis explain the nega- IgE is commonly related to bad clinical prognosis, and tive correlation between the time of congenital infection IgG4 could have either negative or no influence.7,22,100,101 and the magnitude of clinical problems. These compo- The few studies on the role of anti-T. gondii antibodies on nents of the immune response mature through gestation congenital infections indicate that fetuses or newborns and, therefore, the conceptus is more capable of protect- who produce specific IgG2, IgG4 or IgE have bad clinical ing him (her)self from the parasite later in pregnancy. prognosis.7,22,100,101 Genetic polymorphisms related to an early Th1 response

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