Host defenses to microbial invasion include the phylogenetically older but rapidly developing antigen-independent or innate immunity and the much more slowly developing specific or adaptive immunity (2, 35, 82, 91). Innate immune responses are triggered by bacteria, viruses, protozoa, and fungi, as non-self, and involve nonspecific activation of neutrophils, monocytes and macrophages, dendritic cells (DCs), natural killer (NK) cells, and complement. The importance of innate immunity in defense against mycobacteria is illustrated by the observation that patients with T-cell-negative, B-cell-negative, and NK cell-positive severe combined immunodeficiency (SCID) may survive inadvertent vaccination with bacillus Calmette-Guérin vaccine (64).
Phagocytosis as a mechanism of innate immune defense has served as the classical model for studying host-parasite interactions, and significant progress has been made toward understanding the molecular mechanisms of phagocytic uptake and microbial killing (19, 25, 54, 57, 59). Recently, Toll-like receptors (TLRs) have emerged as central points of innate immunity (82, 91). TLRs represent a conserved family of immune receptor sensing molecules on a wide variety of pathogens. These receptors recognize pathogen-associated molecular patterns, which results in activation of NF-κB and other transcription factors including interferon (IFN) regulatory factors. TLRs are expressed on the surface of monocytes, macrophages, DCs, and epithelial cells or in the cytoplasm of cells from different tissues. Other immune receptors involved in innate immune responses are the macrophage mannose receptor (MR) and dectin-1 (25, 93). Ligand binding to innate receptors generates intracellular signals, initiates gene activation, and enhances the release of cytokines and chemokines at the site of immune activation. Chemokines recruit innate immune effector cells such as granulocytes, monocytes, macrophages, and NK cells (32, 63, 65). An important humoral component of innate immunity is the complement system, which can be activated through the alternative and lectin pathways, in addition to the classical pathway, leading to antibody-independent opsonization and opsonophagocytosis (55, 75).
Innate immunity is ontogenetically older than adaptive immunity, but innate recognition of pathogens is the first step in inducing adaptive immunity (35). In vertebrates, innate and adaptive immunity are overlapping and intervening. One major difference in the biology of the two systems is that several responses for innate immune recognition are encoded in the germ line DNA and, in contrast to adaptive immune responses, do not require gene rearrangement (35).
NEONATAL INNATE IMMUNITY
Human neonates and young infants are more vulnerable to infectious agents than older children and adults and are especially susceptible to infections with intracellular pathogens. Some of the pathogens causing infections in utero, intrapartum, and postpartum evoke fetal and neonatal innate immune responses. These agents include group B streptococci (GBS), Escherichia coli, Listeria monocytogenes, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), respiratory syncytial virus (RSV), Toxoplasma gondii, and Candida species. Innate immunity against these pathogens represents the critical first-line barrier of host defenses, as newborns have a naïve adaptive immune system. The past decade has brought great strides in our understanding of innate immune mechanisms in humans. An increasing body of evidence suggests that neonatal innate responses may not be fully developed, allowing early dissemination of infections. This review describes recent advances and current understanding of innate neonatal immunity to infectious agents that are thought to be responsible for significant morbidity and mortality in newborns. Neonatal infection by sexually transmitted disease pathogens (Treponema pallidum, Neisseria gonorrheae, and Chlamydia trachomatis), human immunodeficiency virus (HIV), and hepatitis viruses will not be discussed here. A better understanding of molecular mechanisms that underlie neonatal immune functions may improve our ability to prevent and treat neonatal infections.
INNATE RESPONSES TO NEONATAL PATHOGENS
GBS.
GBS is the foremost cause of neonatal bacterial infections, and mortality of invasive GBS disease in newborns remains high despite advances in intensive care and susceptibility of the pathogen to penicillin and gentamicin (16, 78). The lack of pathogen-specific maternal antibodies to the capsular polysaccharide antigen was supposed to contribute to susceptibility and the severe course of disease in newborns. Accordingly, it was believed that effective vaccination would be a way to reduce the incidence of GBS disease over the long term. In a study including 321 healthy term newborns, immunoglobulin G (IgG) antibodies against capsular polysaccharides of GBS serotypes Ia, II, and III were present in 98 to 100% of cord sera (10). However, the concentrations of IgG antibodies were often low and might not have been sufficient for protection. Naturally occurring IgG antibodies with the capacity to opsonize GBS type III in a complement-dependent manner may also play a role in host defense against these pathogens (22).
Neutrophils are the predominant mobile phagocytes of circulating blood and may contribute to killing of GBS even more than mononuclear phagocytes do. Importantly, exposure to recombinant human IFN-γ was found to activate cord blood neutrophils and to result in enhanced chemotaxis and increased concentrations of free intracellular calcium (31). These data suggest that IFN-γ may enhance the newborns' own host defense by activating neutrophils.
Monocytes and macrophages have a rich diversity of cell surface receptors complementing the diversity of microbial molecules that they are likely to encounter, often in the context of soluble opsonins such as complement and antibodies. Earlier studies showed that the capacity of cord blood monocytes to kill serum-opsonized GBS type III was decreased compared to the capacity of adult blood monocytes (56). Interactions between serum-opsonized GBS and monocyte-derived macrophages isolated from cord blood were also studied by using resident and cytokine-activated cells (53). These results showed that resident cord and adult macrophages efficiently phagocytosed serum-opsonized GBS, but the ingested bacteria survived inside the cells. Bacterial killing by cord macrophages was augmented by granulocyte-macrophage colony-stimulating factor but not by IFN-γ, suggesting differential modulation of bacterial killing by these cytokines. Survival of GBS in neonatal macrophages provides an additional explanation to the severity of GBS disease in early life.
GBS types Ia and III may impair microbicidal systems in murine macrophages by inhibiting protein kinase C-dependent signal transduction pathways (17). Alternatively, macrophages may fail to kill GBS unless they are activated. After phagocytosis, these cells may become permissive for bacterial replication. Therefore, ingestion by macrophages of opsonized GBS may not only enhance but also interfere with elimination of these bacteria at the site of tissue infection.
GBS vigorously activates inflammatory cytokine responses by innate immune cells (36). Impaired interleukin-12 (IL-12) production by GBS-exposed mononuclear phagocytes has recently been proposed to be linked to IFN-γ deficiency in newborns. GBS-stimulated mRNA accumulation and protein secretion of both IFN-γ and IL-12 in mononuclear cells from cord and adult blood were studied (36). By using reverse transcriptase PCR and quantitative densitometry assays, the kinetics of GBS-stimulated accumulation of IFN-γ mRNA and IL-12 mRNA were compared in cord and adult cells. After 12 to 18 h of incubation, significantly decreased mRNA accumulation for both IFN-γ and IL-12 was detected in cord cells compared to adults. The concentrations of IFN-γ and IL-12 in suspensions of GBS-exposed cord mononuclear cells were also significantly lower than in adults at 12 and 18 h. These data suggest that, in addition to lymphocyte immaturity, IFN-γ deficiency in neonates may be linked to decreased production of IL-12 by cord mononuclear phagocytes, at least when these cells are stimulated with GBS. This observation also suggests that strategies to enhance neonatal host defense against GBS may include administration of IL-12.
The capsule of GBS is well characterized as one of the virulence factors of streptococci. The capsule protects GBS from opsonization by C3 through inhibition of the alternative complement pathway in the absence of type-specific antibodies (76). In addition, streptococcal proteins localized to the surface of bacteria may bind complement factor H, retaining its ability to down-regulate complement activation (6). Recently, a surface-localized protease, CspA, that may play an important role in GBS pathogenesis as an antiphagocytic surface factor was described (28). CspA was found to be required for GBS cleavage of human fibrinogen. GBS mutants that failed to express cspA, the gene coding for CspA, displayed a significantly decreased virulence in a neonatal rat model of GBS infection and an increased sensitivity to opsonophagocytic killing. Further characterization of the expression and function of surface-localized GBS proteins and enzymes will help us to understand better how GBS that evade the host innate immune response cause severe infections in newborns.
E. coli.
E. coli is one of the leading gram-negative bacteria that cause neonatal meningitis and sepsis (84). The mortality rate and the neurological squeal remain high despite advances in antimicrobial therapy. Intracellular survival of E. coli represents one important pathogenicity mechanism. E. coli K1, which causes meningitis in neonates, is able to enter and survive in human macrophages and peripheral blood monocytes (85). Outer membrane protein A (OmpA) expression on the surface of bacteria plays an important role in binding to and phagocytosis by macrophages in the absence of opsonization. E. coli expressing OmpA is able to bind the classical complement fluid-phase regulator C4b-binding protein to avoid deposition of C3 and C5, subsequent phagocytosis by granulocytes, and activation of the membrane attack complex (69). In addition, IgG does not bind efficiently to the surface of E. coli K1, allowing the bacteria to avoid recognition via the Fcγ receptors of granulocytes. Deficiency of the alternative complement pathway in cord blood contributes further to the opsonic defect in neonates against E. coli (55). Under such conditions, entry and survival within macrophages could play an important role in the development of bacteremia and the course of meningeal infection by E. coli (85).
Neonatal innate immune cells are characterized by decreased responses to pathogen-derived or physiologic stimuli like lipopolysaccharide (LPS) and IFN-γ, respectively (44, 106). LPS, the primary constituent of E. coli and other gram-negative bacteria, induces inflammation by binding to the TLR4/MD2/CD14 complex on macrophages (35). Recent research has shown normal expression of both TLR-4 and CD14 molecules on cord blood mononuclear cells but decreased TLR-4-mediated signaling and ligand-induced tumor necrosis factor α (TNF-α) release by these cells exposed to LPS (44, 106). These data suggest that the risk of overwhelming infection by E. coli in human neonates may be related to impaired TLR-4-mediated responses by macrophages, in addition to decreased opsonophagocytosis. Other authors found that neonatal mononuclear cells produce an enhanced amount of TNF-α in response to LPS or GBS (102). These findings are in concert with the enhanced sensitivity of neonates to TNF-induced shock.
Listeria.
Listeria infection in humans occurs most commonly in newborns and in immunosuppressed children and adults (45). Perinatal infections caused principally by L. monocytogenes are usually secondary to maternal infection or colonization. Macrophage activation is critically important for an efficient killing of Listeria, and macrophage activation in vivo by IFN-γ is a sine qua non for protection (9, 46). The effectiveness of innate immunity in host defense against Listeria has been exemplified by studies using SCID mice that lack both T-cell and B-cell immunity. These mice were remarkably resistant to infection with L. monocytogenes due to a rapid neutrophil response followed by activation of macrophages and were able to control infection for several days (18). However, listeriosis in mice with an SCID mutation results in a chronic infection characterized by abundant granulomas, microabscesses, and neutrophil infiltrates occurring mostly in the liver (11). Therefore, even though the innate immunity is effective to provide protection, an adequate immune response, i.e., clearance of bacteria, granuloma formation with lymphocytes, and disappearance of microabscesses, requires specific immunity. Adoptive transfer studies showed a decisive role of CD4+ and CD8+ T cells in augmenting innate antibacterial host defenses and ensuring long-term survival of Listeria-infected adult mice (11).
The capacity of CpG oligodeoxynucleotides (ODN) to stimulate protective immune responses to Listeria was recently studied in newborn mice (34). These studies showed that DCs, macrophages, and B cells from 3-day-old mice responded to CpG stimulation by secreting IFN-γ, IL-12, and TNF-α. In addition, spleen cells from CpG-treated newborn mice produced large amounts of cytokines and nitric oxide when exposed to Listeria in vitro. In concert with these findings, newborns treated with CpG ODN were protected from lethal Listeria challenge (34). These data suggest that cellular elements of the neonatal immune system, similar to those of adult mice, may respond to stimulation by CpG ODN, thereby reducing host susceptibility to infectious pathogens.
The hematopoietic growth factor Flt3 ligand (FL) was found to induce a >100-fold increase in the innate resistance to Listeria infection in neonatal mice (95). In particular, FL induced increases in DC numbers as well as IL-12 production by these cells (96). The increased IL-12 production may be crucial in defense against Listeria in vivo through stimulating IFN-γ release by T cells and NK cells and most likely explains the increased survival of FL-treated neonatal mice. Although these studies did not clearly define differential responses to FL by adult versus neonatal mice, they indicate that newborn mice treated with this hematopoietic growth factor have a distinct advantage over untreated littermates to control Listeria infections.
TLRs (TLR-2, TLR-4, and TLR-5) have been implicated in mice and humans as signaling receptors for L. monocytogenes (23, 80, 94, 98). Studies in mice showed that TLR-2 plays a critical role in controlling Listeria infection (94). In particular, TLR-2-deficient mice were more susceptible to systemic infection by Listeria than were wild-type mice, with a reduced survival rate and an increased bacterial burden in the liver.
HSV.
HSV is a formidable pathogen causing disseminated or central nervous system disease with a high mortality rate in the first weeks of life (101). Infection is acquired during the birth process as the neonate comes in contact with the virus during passage through an infected birth canal or through contact with individuals with active HSV lesions. Cellular immune responses mediated by T cells are impaired in newborns compared with older children and adults, which may be responsible for rapid progression of the disease (14, 86). Recent studies have shown that both HSV-1 and HSV-2 induced secretion of IL-6 and IL-8 from adult peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner (41). In addition, HSV-1 and HSV-2 activated NF-κB in TLR-2-transfected HEK 293 cells but not control HEK cells or TLR-4-transfected HEK cells (40). Analysis of IL-6 and IL-8 responses revealed that cord blood cells produced significantly higher amounts of these cytokines in response to stimulation with HSV-1 than did adult blood cells (40). These findings are in concert with previously published data indicating that term and preterm infants produce enhanced IL-6 and IL-8 and that clinical manifestation of HSV infection is associated with increased production of inflammatory cytokines (79). Nevertheless, the link between in vitro and in vivo data is only indirect, and further research is needed to determine whether ongoing overproduction of inflammatory cytokines is a consistent component of HSV pathology in newborns.
The effect of FL on neonatal innate immunity to HSV infection has recently been studied in mice (95). After FL treatment, the nature and quality of resistance were analyzed for short-term innate effect and for survival of neonatal mice. Data showed that FL induced an IFN-α/β-associated immune response in newborn animals by expanding cells of the DC linage. A significant number of mice lacking mature T and B cells died after challenge with HSV-1, whereas 30 to 40% of FL-treated mice survived HSV-1 infection for more than 21 days (95). This observation indicated that innate immunity was decisive in defense against HSV and that manipulation of the innate immune system by cytokine treatment may provide a tool to improve clinical outcomes of neonatal HSV infection.
CMV.
CMV is the most common cause of intrauterine infection, affecting 0.3 to 2.2% of live-born infants (4). Congenital CMV infection is a leading cause of sensorineural hearing loss, cognitive and visual impairment, and cerebral palsy. The virus can be transmitted to the fetus during primary maternal infection in pregnancy, but it can also be transmitted even when maternal infection occurred years prior to conception (24, 81). Earlier studies suggested that the increased susceptibility of the fetus to CMV infection could be related to defective cell-mediated immunity (26, 67). Recently, the presence of functionally mature cytolytic CD8+ T lymphocytes in newborns with congenital CMV infection was reported (47). This finding suggests that intrauterine antigen stimulation has the potential to elicit protective immunity in the fetus and that, in contrast to CD4+ T cells, the expression of efficient CD8+ effector function in newborns may be preserved. Pertinent to this finding, functionally mature CD8+ cytotoxic responses were documented in infants during primary infection with RSV (61). The overall efficiency of CD8-dependent T-cell function in fetal or neonatal life, however, remains unclear. Evidence suggests that neonatal CD4+ T cells are deficient in activation-associated intracellular signaling and require high levels of costimulation to achieve maximal activation (2, 27, 96). In this regard it is noteworthy that CD4+ T cells play an essential role in promoting the long-term activation and terminal differentiation of CD8+ T cells and in reactivation of CD8+ memory cells.
CMV, as a cofactor, may be involved in the pathogenesis of HIV infection and AIDS (72). A cohort-based prospective study was performed to examine the possible association of CMV infection with the progression of HIV disease in infants who were born to HIV-1-infected women and whose CMV status was known (38). At birth, the frequency of CMV infection in HIV-1-infected infants and in infants not infected with HIV-1 was 4.3% and 4.5%, respectively, which was higher than the rates of 0.3 to 2.2% in the general population. However, at 6 months of age, CMV infection was diagnosed in 39.9% of HIV-1-infected infants and in only 15.3% of noninfected infants. The cumulative rates of CMV infection over a period of 48 months remained significantly higher among HIV-1-infected children, and the rate of CMV transmission from mothers to offspring was especially high during the first 12 months (38). These data suggest that HIV-1-infected children have a higher rate of CMV infection acquired postnatally and that CMV infection is associated with an increased risk of HIV-1 disease progression. It is likely that CMV and HIV-1, two immunosuppressive viruses, may act synergistically to accelerate disease progression. Whatever the mechanism, these observations suggest that strategies to prevent vertical and horizontal CMV infection in HIV-1-infected infants and children should be applied in order to decrease and prolong disease progression and central nervous system disease.
EBV.
EBV infection occurring in early childhood is usually not associated with any defined clinical disease (87). However, if primary infection is delayed until adolescence or adulthood, a high proportion of affected individuals develop infectious mononucleosis (IM), characterized by increased numbers of EBV-infected B cells in the peripheral blood and massive oligoclonal expansion of EBV-specific CD8+ T cells (88). IM can be expected to occur when primary EBV infection is not adequately controlled, leading to a subsequent overstimulation of CD8+ T cells by EBV-infected B cells. This concept is in agreement with fulminant IM occurring in patients with X-linked lymphoproliferative disease, an inherited immune deficiency characterized by defective immune responses to EBV infection. However, this would also imply that EBV infection may be controlled better in newborns and infants than in adolescents or adults. Recently, there have been a number of studies of CD4+ T cells, which are able to inhibit EBV-transformed lymphoblastoid B cell line growth (89, 103). These transformed B cells can activate CD4+ T cells and NK cells from both adult and fetal blood. Differences in the activities of CD4+ T cells and NK cells may not explain the immunological and clinical phenotypes of EBV infection in different age groups. However, CD8+ T-cell responses to EBV-infected B cells may be weaker in newborns and infants, explaining the lack of clinical manifestation of infection in early life.
VZV.
VZV may cause significant morbidity and mortality in fetuses and newborns, and vaccinating VZV-susceptible women prior to pregnancy can prevent both vertical and horizontal transmission of varicella, suggesting a role for antibody-mediated immunity (68). Fetal varicella syndrome arises in about 2% of cases of maternal varicella, occurring during the first 20 weeks of gestation (68, 70). VZV infection in newborns may result from either vertical or horizontal transmission. Perinatally acquired varicella occurs mostly after the onset of maternal viremia but before maternal antibody develops. Visceral organ involvement and a high mortality rate are characteristic features of perinatal varicella (13). Innate immunity in the “antibody-free” window period is therefore critical to control infection. PBMCs from adults were found to produce a large amount of IFN-γ in response to VZV antigen, suggesting that a Th1 response with IFN-γ production may be important in early host defense against VZV (7, 30). Remarkably, VZV did not drive cord blood mononuclear cells (MC) to release significant IFN-γ production (107). A real-time reverse-transcription PCR analysis of IFN-γ mRNA expression showed that VZV induced a significantly higher IFN-γ mRNA in PBMCs than in cord blood MC. IFN-γ production is regulated by T-bet expression mediated by STAT-1 (signal transducer and activator of transcription 1) (3, 73). Recent data suggested that VZV did not upregulate T-bet mRNA significantly in cord blood MC in contrast to its effect in adult PBMCs. These data indicate a poor Th1 response and an impaired innate immune response to VZV in neonates.
RSV.
RSV infection is one of the most common human viral diseases worldwide, and virtually every child is infected by the third birthday (66). The virus does not normally replicate outside of the bronchopulmonary tree, and the infection is exquisitely restricted to the respiratory mucosa. RSV proteins such as the major surface glycoprotein (G) and the fusion (F) protein, which is a large envelope glycoprotein, are essential for viral attachment and penetration, respectively, and are important in initiating immune responses (66, 99). Both G and F glycoproteins are able to induce neutralizing antibody responses and long-term immunity. However, in young infants antibody-mediated immunity might contribute to lung pathology as well. Despite the presence through the first few months of life of maternal antiviral antibodies passively transferred to the fetus, prevalence of more severe forms of RSV disease is greatest in young infants. In a cohort of infants, not only did maternal neutralizing antibodies fail to prevent infection with RSV, but also the severity of pneumonia was inversely related to the level of neutralizing antibodies, an intriguing observation as far as passive neonatal immunity is concerned (39, 42). In contrast, administration of RSV-specific immunoglobulin or monoclonal antibody preparations to high-risk infants may prevent bronchiolitis and hospitalization (33, 71). These data clearly indicate that further research is needed to define the role of specific antibodies in antiviral immunity in RSV disease in early life.
Several reports suggested detectable innate cytokine responses to RSV at birth. In vitro, both cord and adult monocyte-derived macrophages exhibited production of high levels of IL-6 and TNF-α within 24 h after viral exposure (60). In contrast to adult cells, little or no production by cord macrophages of these cytokines was observed 24 h after exposure to live RSV. These data indicated that neonatal macrophages may be less efficient in a sustained induction of inflammatory cytokine production. Others found that cord mononuclear cells showed no proliferation response to exposure to inactivated RSV and, when exposed to live virus, produced fewer innate and no adaptive cytokines (39). The major difference in cytokine responses of cord and adult mononuclear cells to RSV exposure appears to be that cord cells produce almost entirely innate cytokines, whereas both innate and adaptive cytokines are produced by adult cells. Consequently, adaptive cytokine responses may be required for an efficient innate immune response to RSV. Humans are born with the capacity to mount innate cytokine responses to RSV, but due to the lack of in utero sensitization, infants may remain highly susceptible to the virus until adaptive cellular immunity develops.
In mice sensitized with recombinant vaccinia virus vector, the G and F glycoproteins differentially regulated cytokine responses (5). Whereas G protein induced a Th2-type response characterized by secretion of IL-4 and IL-5, F protein induced the secretion of IL-2 and IFN-γ. In addition to inflammatory and immunoregulatory cytokines, chemokines are also induced in the respiratory tract after natural RSV infection. Studies of children with RSV bronchiolitis have shown an increased production of chemokines including CXCL8, CXCL5, CXCL3, and CXCL2 in the upper respiratory tract (32, 65). Intriguing recent findings on chemokine production in the lower respiratory tract in infants with RSV bronchiolitis were reported (63). CXC chemokines (CXCL10 and CXCL8) were found to be the most abundant, but CC chemokines (CCL2 and CCL3) were also present. Remarkably, CXCL10, one of the few chemokines capable of binding receptors from different classes (both CXCR3 and CCR3), was present in very large quantity in the RSV-infected lung. Whether chemokine responses are protective or contribute to the pathogenesis of RSV disease needs to be determined. Further clinical studies are required to discover whether chemokine responses induced by RSV occur in other viral or respiratory tract infections in children. Precise elucidation of the role of chemokines in the pathogenesis of RSV bronchiolitis has potentially therapeutic implications because a number of chemokine receptor antagonists are in development.
The clinical spectrum of RSV disease is extremely variable, ranging from mild upper-respiratory tract disease to severe respiratory distress (66). It is likely that genetic heterogeneity contributes to disease severity in addition to known risk factors including prematurity, congenital heart anomaly, and chronic lung disease. Efficient host immune responses to viral pathogens are mediated by Th1 cytokines. As the production of Th1 cytokines can be inhibited by cytokines secreted by Th2 lymphocytes, an adequate balance of Th1 and Th2 cytokines is essential for the efficient eradication of RSV. Several studies have shown a correlation between predominant Th2 responses in infants with RSV disease severity (1, 12, 74). TLR-4 and CD14 have been shown to sense RSV, and TLR-4-deficient mice developed delayed clearance of RSV as well as a predominant Th2 response which correlated with disease progression (29, 92). An association between TLR-4 mutations (Asp299Gly and Thr394Ile) and severe RSV disease has recently been reported, whereas no association between CD14 polymorphisms and RSV bronchiolitis was found (92).
Toxoplasma.
T. gondii, an obligate intracellular pathogen, causes subclinical chronic infections in humans, where it is also an important opportunistic pathogen (62). The human fetus and newborn are especially susceptible to infection by T. gondii. The placenta may act as a barrier to transplacental transmission of parasites from mother to fetus, which occurs mostly in the third trimester. In congenitally infected newborns, infections with T. gondii may result in fibrous or calcified cerebral lesions or ocular lesions that threaten vision (62). However, disseminated infection is rare in congenitally infected infants (20). This may be explained by the unique process of gliding motility that is used by T. gondii organisms to actively invade their vertebral host cells (100). Calcium-mediated protein secretion and MIC2, a thrombospondin-related protein that serves as adhesion for T. gondii, have been implicated in the process of gliding motility. By using this invasion strategy, Toxoplasma escapes phagocytic uptake, and the host cell plays little role in controlling the entry of the parasite. During penetration of host cells, T. gondii restricts access of host cell proteins to the vacuole, thus creating a fusion-incompetent vacuole that lies segregated from the endocytic network (100). This unique intracellular lifestyle provides protection from host surveillance.
Defense against T. gondii infection is mediated primarily by cellular responses involving killing by macrophages and cytotoxic T cells and release of inflammatory cytokines that help infected cells to kill the parasite or to maintain it in a quiescent stage. Cellular immunity is mainly targeted to infected cells that express peptides from the parasite (21). Killing of Toxoplasma by and the survival and replication of this parasite in resident mononuclear phagocytes from newborns and adults, respectively, are comparable (104, 105). In the immune mechanism through which acute T. gondii infection is controlled, IFN-γ plays a central role as a strong activator of resident macrophages to limit intracellular growth of tachyzoites. In vitro studies showed that GTPases are required for IFN-γ-induced suppression of T. gondii growth in macrophages (15). In particular, a 47-kDa protein that possesses inherent GTPase activity and binds to the endoplasmic reticulum and Golgi was found to regulate host resistance to T. gondii through its ability to inhibit parasite growth within the macrophage. In human newborns, both generation of IFN-γ and response to IFN-γ by mononuclear phagocytes are impaired (49, 104). This age-related deficiency is likely to be one critical factor responsible for the increased susceptibility of newborns to T. gondii infection.
Candida.
Infections by Candida are the most common of the fungal infections in newborns (8, 83). Body surfaces are colonized at birth by Candida species residing in the birth canal. Overgrowth of colonizing Candida may lead to mucosal or mucocutaneous candidiasis. The role of passively acquired humoral antibody in defense against invasive candidal disease appears to be negligible in newborns (48). The unique susceptibility to oropharyngeal candidiasis during the first weeks results most likely from the down-regulation of Th1 responses (43, 48, 49).
Invasive candidal disease in neonates is a life-threatening condition which may be explained by developmental deficiencies in the newborn's innate immune system (52). Candida albicans is part of the common commensal of the gastrointestinal tract and invasive candidal disease can arise from entero-circular translocation of the gut flora (58, 77). B cell knockout mice, which lack functional antibodies, are as resistant to mucosal or invasive candidiasis of endogen origin as are immunocompetent controls (97). In addition, patients with X-linked agammaglobulinemia or severe hypogammaglobulinemia do not exhibit an increased susceptibility to either mucocutaneous or invasive candidal infections (52). In such patients T cells and innate immune cells ensure defense against Candida, and macrophages can prevent candidal invasion by phagocytosis and killing of nonopsonized yeasts through innate immune receptors (19, 25, 54, 90).
Recognition and uptake of Candida yeasts involve the macrophage MR, which is a type I membrane protein with three types of domains in the extracellular region (19, 54, 59, 93). Well-characterized lectin activities of the MR are mediated by the cysteine-rich domain which can recognize sulfated sugars, whereas mannose recognition takes place through the C-type lectin-like domains. The extent of phagocytosis and killing of nonopsonized Candida organisms by resident monocyte-derived macrophages were comparable in newborns and adults, and both mannan and mannose-bovin serum albumin complex inhibited ingestion in a concentration-dependent manner, suggesting a role for the MR (50, 52). Exposure of adult macrophages to IFN-γ (up to a concentration 100 U/ml) resulted in increased phagocytosis and killing. In contrast, no enhancement with cord macrophages could be detected under the same experimental condition, and at a concentration of 500 U/ml IFN-γ there was still significantly lower killing and superoxide release by cord macrophages compared to adult cells (57). These data suggested that neonatal macrophages have a normal capacity to ingest and kill Candida through the MR but cannot be fully activated by IFN-γ, a finding that could not be attributed to lower expression or binding to its ligand of IFN-γ receptor on neonatal cells. Remarkably, in response to IFN-γ, a significantly decreased STAT-1 phosphorylation was detected in neonatal cells, suggesting the possibility of negative regulation of IFN-γ receptor signaling in newborns (51). The precise mechanism by which signaling through innate immune receptors may be down-modulated in neonates remains unclear.
Dectin-1, the receptor for binding fungal-derived β-glucan by macrophages and neutrophils, is a small type II transmembrane protein containing one lectin-like carbohydrate recognition domain (25). This receptor can recognize live Saccharomyces cerevisiae and, to a lesser extent, C. albicans. It was previously reported that ligand binding to the MR is not coupled to the activation of the respiratory burst oxydase and superoxide release in macrophages, in contrast to binding antibody-opsonized Candida to Fc receptors on these cells (54). The effect of ligand binding to dectin-1 on the respiratory burst activity has not been studied. However, a TNF-α response was generated by macrophages upon exposure to β-glucan. Remarkably, the production of TNF-α was significantly greater when macrophages were exposed to S. cerevisiae than to C. albicans. This observation is in concert with published data that β-glucan is buried within the cell wall of C. albicans and that Candida uptake by macrophages and keratinocytes can be inhibited by mannan and, to a lower extent, by glucan (37, 54, 90). It is also possible that S. cerevisiae may have a higher density of β-glucan exposed on its surface compared to that of C. albicans.
SUMMARY AND CONCLUSION
Human neonates are highly susceptible to infection by a wide range of bacteria, viruses, protozoa, and fungi. The heightened susceptibility and the severe course of infections in early life can be attributed, at least in part, to the lack of preexisting immunological memory and competent adaptive immunity. In addition, a large body of evidence suggests that several innate immune mechanisms are impaired in neonates. It is also clear, however, that neonates are immunocompetent to mount mature innate as well as adaptive immune responses like nonopsonic uptake of fungi or, under certain circumstances, adult-level T-cell responses. Thus, the challenge of future research will include the discovery of mechanisms that underlie differential immune responses in newborns so that prevention and treatment of neonatal infections can more safely be targeted.