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The present investigation aimed to determine to what extent maternal helminth infection primes parasite-specific cellular responsiveness in neonates. Umbilical cord mononuclear blood cells (UCBC) and peripheral blood mononuclear cells (PBMC) from mothers proliferated in response to mitogenic stimulation with concanavalin A, as well as to bacterial Streptococcus pyogenes-derived (streptolysin O) and helminth-specific antigens of Necator americanus and Onchocerca volvulus. Cellular responses to Echinococcus multilocularis (Em) and Oesophagostomum bifurcum (Oes), helminth parasites not endemic in the study area, were absent (for Em) or very low (for Oes due to antigenic cross-reactivity). Cellular responsiveness to mitogen and antigens was higher in mothers than in their neonates. Several Th1-type (IL-2, IL-12, and IFN-γ) and Th2-type (IL-5 and IL-10) cytokines were produced by UCBC from neonates and PBMC from mothers. Low levels of IFN-γ were elicited by UCBC in response to helminth and bacterial antigens, while secretion of IL-2 was pronounced and similarly high in neonates and their mothers. Amounts of IL-5 produced by UCBC in response to bacterial SL-O and mitogenic stimulation (PHA) were low, but equivalent levels of IL-5 were induced by intestinal helminth and filaria-derived antigens in neonates and mothers. A pronounced production of IL-10 and IL-12 by UCBC was observed – spontaneous IL-10 and IL-12 secretion by UCBC was higher in neonates than by PBMC from mothers. Net amounts of IL-10 elicited by helminth antigens were similar, while net IL-12 in response to mitogen, and bacterial and helminth antigens was significantly higher in mothers than their offspring. Our results indicate that human maternal helminth infection does sensitize in utero for parasite-specific cellular responsiveness in offspring, and also activates specific production of several cytokines, and such children do not present a dominant expression of immunity of either Th1 or Th2.
There is increasing evidence that prenatal T-cell priming occurs via transplacental exposure to antigens and allergens, and such primary immune sensitization may affect the appropriate maturation of the postnatal immune system ( 1–3). In areas where helminth infection is endemic, pregnant women are frequently infected with tissue-dwelling and intestinal helminth parasites, and the transfer of parasite-specific antibodies, the leakage of parasite-specific antigens, and the migration of entire parasites across the placenta may sensitize offspring prenatally ( 4). In utero exposure to pathogen-derived antigens or even entire parasites has been considered a risk factor in offspring for enhanced susceptibility to infection ( 2, 5) and higher parasite densities after exposure in later life ( 6), and prenatal immune priming may contribute to the heterogeneity of host immunity and disease expression, as observed with helminth infections in native populations ( 7). It has previously been shown that prenatal allergic sensitization to parasite antigens occurs in the offspring of filaria-infected mothers ( 8), and that prenatal exposure to allergens may bias T-cell responses toward a Th2-like phenotype ( 3). At the same time, evidence from epidemiologic and experimental studies suggests that prenatal exposure to parasite antigens may protect from disease upon infection in later life ( 9, 10). In general, helminth infections induce Th2-type immune responses that confer, in experimental models, host protection and expulsion of intestinal nematodes ( 11). Similarly, filarial parasites selectively induce Th2-type responses, but such dominant reactivity contributes to immunopathologic symptoms, such as filaria-induced airway hyperresponsiveness ( 12). The pathogenesis of disease caused by Schistosoma infection also appears to be a Th2-mediated process, while Th1-type cytokines reduce immunopathologic disorder and promote protective immunity ( 13). From these investigations, we may conclude that pregnancy, by its selective induction of a Th2-promoting environment at the prenatal age, leading to prenatal sensitization and subsequent activation of Th2-type responses in neonates, may support protective immunity against intestinal helminth parasites, but such expression of immunity may also predispose to chronic infections and immunopatho-genesis in filariasis and schistosomiasis. A large body of evidence, however, indicates that the dominance of either type of immunity may depend on the nature of the parasite and the specific tissues involved in the inflammatory response.
This study aimed to investigate to which extent maternal intestinal helminth infections have sensitized or biased immune responsiveness in neonates toward a Th1- or Th2-like phenotype. Our results indicate that helminth infections in pregnant women do sensitize their offspring in utero for parasite-specific cellular responsiveness, and also activate parasite-specific production of several Th1- and Th2-type cytokines.
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There is increasing interest in the concept that parasite infections during pregnancy and transplacental immune priming may predispose offspring not only to increased susceptibility to chronic infection and parasite per-sistence but also to inappropriate development and expression of immunity in later life ( 5, 18–20). Further-more, deviated or skewed immunocompetence in children of infected mothers may reduce the success of vaccination against infectious and parasite diseases in infants ( 21). This investigation presents substantial evidence that intestinal helminth infections during pregnancy do sensitize offspring in utero for parasite-specific immune responsiveness, and induce cellular production of several Th1- and Th2-type cytokines, but prenatal sensitization did not bias expression of immun-ity toward a dominant Th1- or Th2-type reactivity.
In utero exposure to filarial parasites has been considered to cause parasite-specific cellular hypo-responsiveness and deficient cytokine production in the offspring of filaria-infected mothers ( 2), such impaired immunocompetence then predisposing child-ren to a higher intensity of infection in later life ( 6). But other studies investigating the effects of in utero exposure to Schistosoma haematobium ( 22) and O. volvulus ( 23) suggested that parasite-specific prenatal immune priming generates cytokine responses similar to those observed in adults, indicating that prenatal exposure does not induce tolerance or altered fetal immunity. Moreover, as observed in the present study, intestinal helminth parasites sensitize in utero offspring of infected mothers for parasite-specific cellular responsiveness, and such sensitization is not skewed toward a dominant Th1 or Th2 cytokine profile. In addition to the spontaneous cellular IL-10 and IL-12 production in neonates, helminth antigens induced substantial secretion of IL-10 and IL-12, as well as IFN-γ and IL-5, by cord-blood cells.
However, we have to consider that the dominance of either T helper type 1 or T helper type 2 parasite-specific immune responses could differ between the various parasites involved with respect to the magnitude of the immune stimulation, and the parasite-induced production of isotypes and cytokines in the mother and therefore also in the baby. Low production of IL-2 by PBMC and UCBC in response to helminth-specific antigens has been reported previously ( 22, 23). IL-2 is an autocrine growth factor and often difficult to measure in antigen-stimulated cell cultures; therefore, IL-2, as determined in this study, may simply reflect the use of the cytokine as it is produced. During pregnancy, the cytokine milieu at the fetomaternal interface is constitutively polarized toward the Th2-like profile, with intrauterine production of high levels of IL-4 and IL-10 ( 24). Such polarization is thought to be an evolutionary adaptation designed to protect the placenta against the toxic effects of Th1-type cytokines, such as IFN-γ, the production of which is a major cause of fetal loss ( 25–27).
However, the concept that Th1-type immunity is incompatible with successful pregnancy ( 24) has been challenged by recent studies indicating that exposure to parasite antigens during pregnancy generated IFN-γ and TNF-α, which control Plasmodium falciparum infection in the placenta ( 28, 29). Neonates developed a Th1-type immune response to Mycobacterium bovis bacillus Calmette-Guérin vaccination ( 30), but when children were sensitized in utero to filariasis and schisto-somiasis they presented with reduced M. tuberculosis-specific IFN-γ production after BCG vaccination, showing that prenatal sensitization biased T-cell immunity away from T helper type 1 responses, which are associated with protection against mycobacterial infection ( 21).
In the present study, transplacental immune priming and sensitization were not observed to antigens from parasites not endemic in our study area, such as E. multilocularis. Moreover, none of the mothers of this study were patently infected by O. volvulus or O. bifurcum, and all were serologically negative for parasite-specific IgG subclasses. However, cells from mothers and UCBC from their babies proliferated in response to O. volvulus- and O. bifurcum-specific antigens, and UCBC secreted several cytokines in response to these parasite antigens as well. Such responsiveness provided evidence of the low-level exposure of mothers to O. volvulus. We hold the biologic and morphologic similarity ( 16), and antigenic cross-reactivity between hookworm and O. bifurcum ( 31) responsible for the low cellular proliferation and weak cytokine responses in mothers and their children to O. bifurcum antigens. Previously, the cellular reactivity of UCBC to helminth antigens (i.e., S. haematobium and Brugia malayi) has been observed in neonates from uninfected mothers ( 22, 32), and such responsiveness was also attributed to cross-reactivity among intestinal helminth antigens that were common within the study population. In hookworm- and onchocerciasis-endemic areas, such as rural West Africa, parasite-specific responsiveness in the neonates of hookworm-free and onchocerciasis-free mothers could be induced by the leakage across the placenta of low or ultralow levels of parasite-derived antigens, these antigens providing the necessary stimulus for T-cell priming, and the cause of TH1- and TH2-like cytokine responses in neonates of mothers exposed to infection. Moreover, in utero exposure to helminth and mycobacterial antigens in uninfected mothers has been reported with adult-level Th1 responses in neonates ( 22, 23).
Baseline proliferation in cord-blood cells has been observed in previous studies ( 23, 32), as well as in the present study, and the continuous transfer of immunologically active proteins (e.g., cytokines, IgG antibodies, and antigens) across the placenta from mother to fetus may have activated such cellular reactivity. Systemic activation of the maternal innate immune system ( 33) and soluble placental products, released into the circulation and potentially transferred across the placenta, may also have stimulated monocyte-derived cytokine production (e.g., IL-12 and TNF-α), and this may account for the observed spontaneous proliferation and cytokine responses in UCBC. The spontaneous and also antigen-driven IL-10 and IL-12 production by UCBC, as observed in the present study, further indicates that there is no clear bias, in either mothers or neonates, toward dominant Th1- or Th2-type immunity.
The mechanisms by which maternal infections sensitize offspring are still not fully understood. Interestingly, neonatal T cells require much lower doses of antigen than adult T cells to become activated, tolerated, or biased toward the TH2 phenotype. Other investigators seeking to determine the influence of maternal filarial or S. mansoni infections ( 22, 34) on neonate immunity have found little evidence of skewed immune responses. The dominance of either type of immunity may thus depend on the nature of the parasite, the specific tissues involved in the inflammatory response, and the extent and intensity of parasite exposure and infection. Early exposure to parasite or vaccine antigens may create a state of selective unres-ponsiveness (tolerance) to the same antigen in adulthood ( 18, 35), or it may bias secondary responses to the Th2 phenotype ( 36, 37). Similarly, allergic responses induced in pregnant women by ultralow levels of inhal-ant allergens will prime their offspring in utero ( 38), transplacental priming by environmental allergens will skew the postnatal immunologic memory toward a Th2 reactivity dominated by high-level IL-10 production ( 3), and reduced IFN-γ secretion in neonates has been related to the subsequent development of atopy ( 39).
However, recent studies have shown that neonatal exposure to antigens could induce both immunity or tolerance, and the key factors determining the end result were the type of the antigen-presenting cell, the dose of antigen, and the type of adjuvant ( 40–42). Experimental studies have shown that neonate T cells respond like those of adults, and both Th1 and Th2 primary effector cell populations are generated in neonatal mice with cytokine levels comparable to those produced by adult cells, but despite a balanced Th1/Th2 primary response, responses are skewed toward Th2 upon secondary challenge in later life ( 43).
These observations suggest that while neonates are fully capable of mounting primary Th1-type responses, such Th1-type reactivity may not be stable, and upon secondary antigen challenge, balanced responses become dominated by Th2. Interestingly, when the neonate's primed immune system encounters the envi-ronment, a secondary exposure to high levels of the relevant antigens will further boost Th2-type immunity; in contrast, low antigen exposure promotes the development of dominant Th1 responses ( 1, 44). These investigations, like our present study, show that an initial in utero priming of the T-cell system with parasite-derived antigens may create a balanced Th1/Th2 reper-toire, and once immunologic memory becomes established the postnatal intensity of parasite transmission and rate of parasite accumulation may comprise those factors which decisively determine expression of immunity toward a Th1 or Th2 dominance.
In summary, our investigation of neonates indicates that prenatal sensitization due to maternal intestinal helminth infection induces Th1- and Th2-like primary responses, and that the postnatal encounter with the outside environment, e.g., the intensity of exposure to the relevant antigens, may be the critical determinant for skewing of the initial T-cell response toward host-protective or parasite-permissive immunity. From our observations, we conclude that cellular hyporesponsiveness and dominance of a Th2-type cytokine profile may be manifested in progeny by an increasing helminth parasite load; i.e., with repeated postnatal infections and continuous accumulation of parasites, a cellular anergy may develop that will facilitate helminth parasite persistence as well as predispose to chronic infection. Better understanding of the effects of maternal parasite infection during pregnancy on immunity in offspring may help us to develop strategies for early infant immunization and determine priorities for future disease control, especially in pregnant women.