Transmission of Toxoplasma gondii from Infected Dendritic Cells to Natural Killer Cells

The obligate intracellular parasite Toxoplasma gondii can actively infect any nucleated cell type, including cells from the immune system. In the present study, we observed that a large number of natural killer (NK) cells were infected by T. gondii early after intraperitoneal inoculation of parasites into C57BL/6 mice. Interestingly, one mechanism of NK cell infection involved NK cell-mediated targeting of infected dendritic cells (DC). Perforin-dependent killing of infected DC led to active egress of infectious parasites that rapidly infected adjacent effector NK cells. Infected NK cells were not efficiently targeted by other NK cells. These results suggest that rapid transfer of T. gondii from infected DC to effector NK cells may contribute to the parasite''s sequestration and shielding from immune recognition shortly after infection.Toxoplasma gondii causes chronic infections in up to one-third of the human population and in animals (22, 31). In healthy individuals, primary T. gondii infection causes relatively mild symptoms, whereas in the immunocompromised patient or in the developing fetus, life-threatening manifestations lead to severe neurological and ocular damage (11, 28, 37). Following oral infection, T. gondii parasites typically pass across restrictive biological barriers and rapidly disseminate (13). In this process, T. gondii actively infects a great variety of cell types, including epithelial cells and blood leukocytes (12, 21). In infected cells, the parasites establish nonfusigenic parasitophorous vacuoles, where they can replicate (27, 32, 38).Natural killer (NK) cells and dendritic cells (DC) are two important cell types of the innate immune system. DC-NK cell interactions are important not only in host defense but also for the development of adaptive immune responses (5, 9). The activation of DC by pathogens leads to cytokine secretion, which activates NK cells, which in turn, via cytokines or by direct cell-cell contact, may determine the adaptive immune responses that follow (9, 29). DC are sensitive to NK cell-mediated lysis in vitro and can be eliminated by NK cells in vivo (4, 6, 17, 19, 33, 43). Viral or bacterial infection of DC can reduce their sensitivity to NK cell-mediated lysis by increasing the expression of classical and nonclassical major histocompatibility complex class I molecules on the cell surface (14, 35, 43).DC and NK cells play critical roles in innate immunity during acute Toxoplasma infection, being early sources of interleukin-12 (IL-12) and gamma interferon (IFN-γ), respectively (16, 20, 24, 34, 40). It has recently been suggested that infected DC, and possibly other leukocytes, can act as Trojan horses, potentiating the dissemination of the parasite from the point of infection to distal parts (8, 26). In the early phase of infection with T. gondii, NK cell recruitment to the site of infection is mediated by CCR5-binding chemokines (24). IFN-γ production by NK cells, induced by IL-12 from infected DC or macrophages, has been suggested to be the primary contribution of NK cells to the host defense against T. gondii (18, 25, 39). It can also drive cytotoxic CD8⁺ T-cell immunity to T. gondii even in the absence of CD4⁺ T cells (7). NK cells can also kill T. gondii-infected target cells (42), and perforin has been demonstrated to be important in protecting mice in the chronic stage of infection (10). In the present study, we investigated NK cell interactions with T. gondii-infected DC and, surprisingly, demonstrated how this interaction leads to T. gondii infection of NK cells.     

The Staphylococcus aureus Lipoprotein SitC Colocalizes with Toll-Like Receptor 2 (TLR2) in Murine Keratinocytes and Elicits Intracellular TLR2 Accumulation

SitC is one of the predominant lipoproteins in Staphylococcus aureus. Recently, SitC was shown to be capable of stimulating Toll-like receptor 2 (TLR2), but the mechanism of TLR2 activation by SitC has not been analyzed in detail so far. In this study, we purified C-terminally His-tagged SitC (SitC-His) from Staphylococcus aureus. SitC-His induced interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) release in human monocytes and also NF-κB activation in TLR2-transfected HEK293 cells, indicating TLR2-specific activation. SitC not only induced a TLR2-dependent release of IL-6 in primary murine keratinocytes (MKs) but also induced intracellular accumulation of TLR2, which was time and concentration dependent. Cy2-labeled SitC-His colocalized specifically with TLR2 in MKs and was also internalized in TLR2 knockout MKs, suggesting a TLR2-independent uptake. Neither activation nor colocalization of SitC-His was observed with TLR4 or Nod2. The results show that the native lipoprotein SitC-His specifically colocalizes with TLR2, is internalized by host cells, induces proinflammatory cytokines, and triggers intracellular accumulation of TLR2.Recognition of intruding pathogens is the first step of host defense. The innate immune response is capable of recognizing microbes and provides a first line of defense to the host. It is mediated in part by pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), that specifically recognize microbe-associated molecular patterns (MAMPs) derived from microorganisms (2). TLR2 has been shown to play a crucial role in the host response to Staphylococcus aureus (47). It appears to be a receptor for various structurally unrelated MAMPs, e.g., lipoproteins (27, 39), glycolipids from spirochetes, lipoarabinomannan from mycobacteria, porins from Neisseria (45), lipoteichoic acid (LTA) (32, 40), and peptidoglycan (PGN) (1, 4, 12, 20, 33). However, an increasing number of studies suggest that bacterial lipoproteins are the major, if not sole, TLR2-activating molecules of Gram-positive bacteria (18, 27, 43, 48, 52).The N termini of bacterial lipoproteins contain a unique S-diacylglyceryl cysteine moiety. In Escherichia coli and other Gram-negative bacteria, as well as in mycobacterial and spirochetal species, an additional acyl group is linked to the amino group of this cysteine (16), resulting in the formation of triacylated membrane anchor structures. Lipoproteins are synthesized as preproteins with a distinct type II signal sequence containing a conserved lipobox consensus motif (28). In the S. aureus genome, more than 50 genes contain the type II signal sequence typical for lipoproteins. Some of them have been annotated as substrate binding components of ABC transporters that are involved in nutrient and iron acquisition, and one of the predominant lipoproteins is SitC (43). SitC is part of the iron transporter SitABC (10).Until now, only synthetic lipoprotein analogs, such as N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cyste- inyl-(lysyl)3-lysine (Pam₃Cys; tripalmitoyl cysteinyl) lipopeptide, Pam₃CSK₄ (adapted from the Escherichia coli Braun''s lipoprotein), and dipalmitoyl MALP-2 (macrophage-activating lipopeptide 2 kDa) from Mycoplasma fermentans (31), have been shown to mimic the proinflammatory properties of bacterial lipoproteins (46). This led to a model in which triacylated lipopeptides signal through TLR2/TLR1 heterodimers, whereas diacylated lipopeptides signal through TLR2/TLR6 heterodimers. Indeed, the structure of TLR1/TLR2 heterodimers with Pam₃CSK₄ suggests that both receptors are involved in binding the lipopeptide (24). On the other hand, stimulation of TLR1- and TLR6-deficient mice with di- and triacylated lipopeptides (e.g., Pam₂CSK₄ and Pam₃CGNNDESNISFKEK) revealed that neither TLR1 nor TLR6 was necessary for stimulation (8).Whether lipoproteins of S. aureus are di- or triacylated is still not clear. Gram-negative bacteria possess an N-acyltransferase (Lnt) that transfers an acyl group to the amino group of the S-diacylated cysteine residue, yielding a triacylated (N-acylated, S-diacylated) lipoprotein. However, an Lnt homolog has not been found in Gram-positive bacteria such as staphylococci. Therefore, it was assumed that the lipoproteins in S. aureus were diacylated. Indeed, N-terminal analysis of an S. aureus lipoprotein (SAOUHSC_02699) revealed that it is diacylated (48). However, it has also been reported that purified SitC (another lipoprotein) is triacylated (27). Although the major role of lipoproteins in TLR2 activation seems to be established, detailed information on molecular lipoproteins interacting with TLR2 in host cells is still unclear.In this study, the interaction of the staphylococcal lipoprotein SitC with TLR2 of primary murine keratinocytes (MKs) was examined. We show that SitC colocalizes specifically with TLR2 and stimulates proinflammatory cytokines and intracellular TLR2 expression.     

γδ T Cells but Not NK Cells Are Essential for Cell-Mediated Immunity against Plasmodium chabaudi Malaria

Blood-stage Plasmodium chabaudi infections are suppressed by antibody-mediated immunity and/or cell-mediated immunity (CMI). To determine the contributions of NK cells and γδ T cells to protective immunity, C57BL/6 (wild-type [WT]) mice and B-cell-deficient (J_H^−/−) mice were infected with P. chabaudi and depleted of NK cells or γδ T cells with monoclonal antibody. The time courses of parasitemia in NK-cell-depleted WT mice and J_H^−/− mice were similar to those of control mice, indicating that deficiencies in NK cells, NKT cells, or CD8⁺ T cells had little effect on parasitemia. In contrast, high levels of noncuring parasitemia occurred in J_H^−/− mice depleted of γδ T cells. Depletion of γδ T cells during chronic parasitemia in B-cell-deficient J_H^−/− mice resulted in an immediate and marked exacerbation of parasitemia, suggesting that γδ T cells have a direct killing effect in vivo on blood-stage parasites. Cytokine analyses revealed that levels of interleukin-10, gamma interferon (IFN-γ), and macrophage chemoattractant protein 1 (MCP-1) in the sera of γδ T-cell-depleted mice were significantly (P < 0.05) decreased compared to hamster immunoglobulin-injected controls, but these cytokine levels were similar in NK-cell-depleted mice and their controls. The time courses of parasitemia in CCR2^−/− and J_H^−/− × CCR2^−/− mice and in their controls were nearly identical, indicating that MCP-1 is not required for the control of parasitemia. Collectively, these data indicate that the suppression of acute P. chabaudi infection by CMI is γδ T cell dependent, is independent of NK cells, and may be attributed to the deficient IFN-γ response seen early in γδ T-cell-depleted mice.Malaria remains a leading cause of morbidity and mortality, annually killing about 2 million people worldwide (32, 33). Despite decades of research, malaria is a reemerging disease because of increasing drug resistance by malarial parasites and insecticide resistance by the mosquito vector. Most infected individuals do not succumb to malaria but develop clinical immunity where parasite replication is controlled to some degree by the immune system without eliciting clinical disease or sterile immunity (14, 38).Understanding the immunologic pathways leading to the control of blood-stage parasite replication is important for defining the mechanisms of disease pathogenesis and improving vaccines currently in development. The early events of the immune response depend upon activation of the innate immune system, which regulates the downstream adaptive immune response needed to control or cure (44). Natural killer (NK) and γδ T cells function early in the immune response to pathogens as components of the innate immune system. Both cell types have been proposed to play significant roles in the subsequent clearance of blood-stage malarial parasites by activating the adaptive immune system (35, 43, 44). The mechanism by which they accomplish this appears to be mediated via their secretion of gamma interferon (IFN-γ) induced by cytokines such as interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-α), and IL-6 produced by other components of the innate immune system, including macrophages and dendritic cells (17, 25, 26, 37, 49).Blood-stage malaria parasites are cleared by mature isotypes of antibodies and/or by antibody-independent but T-cell-dependent mechanisms of immunity (2, 15, 22). Both responses require CD4⁺ αβ T cells; in addition, the expression of cell-mediated immunity (CMI) during both acute and chronic malaria is dependent on γδ T cells activated by CD4⁺ αβ T cells (29, 47, 49, 50). Wild-type (WT) mice depleted of γδ T cells by antibody treatment or gene knockout suppress P. chabaudi parasitemia by antibody-mediated immunity (AMI) (21, 52). Mice depleted of B cells by the same procedures also cure their acute infections in the same timeframe as intact control mice but then develop chronic low-grade parasitemia of long-lasting duration, indicating that B cells and their antibodies are needed to sterilize the infection as we originally reported (15, 48) and has since been confirmed by others (51). B-cell-deficient mice depleted of γδ T cells cannot suppress P. chabaudi parasitemia (49, 50, 52).The prominent role played by IFN-γ in immunity to malaria is generally accepted by most researchers. P. chabaudi malaria is more severe in WT mice treated with neutralizing antibody and in IFN-γ^−/− mice, as indicated by the increased magnitude and duration of parasitemia and mortality in mice deficient in IFN-γ versus intact controls (24, 39, 46). In B-cell-deficient animals, the similar neutralization of IFN-γ by treatment with anti-IFN-γ monoclonal antibody (MAb) or gene knockout of IFN-γ has an even greater effect on the time course of parasitemia, which remains at high levels and fails to cure (1, 46), indicating that IFN-γ is essential for the expression of anti-parasite CMI and contributes to AMI in this model system.The early source of IFN-γ remains controversial, with both NK cells and γδ T cells being proposed to produce this critical cytokine necessary for the activation of the adaptive immune response and the development of protective immunity (9). The results of earlier genetic studies failed to correlate susceptibility to P. chabaudi infection with NK activity (31, 44). Subsequently, Mohan et al. (25) reported that NK cell activity against tumor cell targets correlates with protection against P. chabaudi; anti-asialo GM1 polyclonal antibody depletion of NK cells results in significantly increased levels of peak parasitemia and a prolonged duration of infection compared to controls. The mode of action by which NK cells function appears to be via the secretion of cytokines (25) rather than direct cytotoxicity against the blood-stage parasites. The surface expression of lysosome-associated membrane protein 1 (LAMP-1) by subsets of human NK cells exposed to Plasmodium falciparum-infected erythrocytes may suggest otherwise (20). NK cells in collaboration with dendritic cells are responsible for optimal IFN-γ production dependent upon IL-12 (17, 36, 39, 40). In contrast to the findings of Mohan et al., other studies indicate similar P. chabaudi parasitemia in depleted mice and intact controls after NK1.1 MAb depletion of NK cells (19, 41, 53). Using microarray analysis of blood cells from P. chabaudi-infected mice, Kim et al. (18) reported a rapid production of IFN-γ and activation of IFN-γ-mediated signaling pathways as early as 8 h after infection; however, NK cells did not express IFN-γ or exhibit IFN-γ-mediated pathways in their analysis. At this time, NK cells are replicating and migrating from the spleen to the blood. In humans with P. falciparum malaria, increased production of IFN-γ by PBMC in response to parasitized RBCs correlates with protection from high-density parasitemia and clinical malaria (10, 11); early IFN-γ production by PBMC obtained from malaria naive donors is primarily by γδ T cells and not by NK cells (26). Animal models by definition do not exactly mimic the human condition, and the experimental malaria in mice uses distinct species from those that infect humans. Nevertheless, analysis of protective immunity provides important information on how a protective immune response to Plasmodium may be elicited.Whether both NK cells and γδ T cells have essential roles during the early stages of the immune response to blood-stage malaria remains to be determined. Likewise, whether these cells function early in CMI to malaria parasites is unknown. To address these issues, we infected NK-cell- or γδ-T-cell-depleted J_H^−/− mice with blood-stage P. chabaudi. The resulting time course of parasitemia was monitored and compared to control mice. In addition, spleen cells from depleted and control mice were profiled by cytofluorimetry, and the serum levels of inflammatory cytokines were measured.     

Natural Killer Cell Dysfunction during Acute Infection with Foot-and-Mouth Disease Virus

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. The role of these cells in foot-and-mouth disease virus (FMDV) infection is unknown. Previously, we characterized the phenotype and function of NK cells from swine (F. N. Toka et al., J. Interferon Cytokine Res. 29:179-192, 2009). In the present study, we report the analysis of NK cells isolated from animals infected with FMDV and tested ex vivo and show that NK-dependent cytotoxic activity against tumor cells as targets was impaired. More relevantly to this infection, the killing of target cells infected with FMDV also was inhibited. Further, the proportion of NK cells capable of producing gamma interferon and storing perforin was reduced. Peripheral blood mononuclear cells isolated from infected animals are not productively infected, but virus exposure in vivo resulted in the significant induction of NKp30 and Toll-like receptor 3 expression and the moderate activation of SOCS3 and interleukin-15 receptor mRNA. However, there was little alteration of mRNA expression from a number of other receptor genes in these cells, including SH2D1B and NKG2A (inhibitory) as well as NKp80, NKp46, and NKG2D (activating). These data indicate that this virus infection influences the ability of NK cells to recognize and eliminate FMDV-infected cells. In addition, a reduction in NK cell cytotoxicity coincided with the increase in virus titers, indicating the virus blocking of NK cell-associated innate responses, albeit temporarily. These effects likely culminate in brief but effective viral immune evasion, allowing the virus to replicate and disseminate within the host.Innate immunity is a vital part of the overall host immune response to invasion by pathogens, particularly during virus infections. Natural killer (NK) cells occupy a critical position in the initial host responses against infection. Originally, NK cells were discovered on the basis of their capability to kill certain tumors without prior activation. Now the role of NK cells has been defined in virus infections such as human cytomegalovirus (8, 11, 55), murine cytomegalovirus virus (2, 32), influenza virus (28, 35), herpes simplex virus (44, 52), ectromelia virus (16, 41), and human immunodeficiency virus (HIV) (14, 50, 56). In two of these infections, a lack or deficiency in NK cell function leads to increased susceptibility to infection (6, 10).The initiation of NK cell responses is thought to originate from signals delivered by the professional pathogen-sensing system, which is comprised mainly of dendritic cells (DC) (21, 47, 57). Although the evidence is not yet definitive, the direct activation of NK cells also may occur through pathogen recognition receptors expressed by NK cells (49, 51). The cross-talk between NK cells and DC leads to the activation of NK cells, after which they operate in a manner that is dependent on the sensing expression of specific molecules induced on virus-infected cells through receptors present on the NK cell surface. In part, the recognition of an infected cell by NK cells relies on the detection of the missing self, i.e., the lack of major histocompatibility complex class I expression on the infected cell surface. Ultimately, the balance between signals from both inhibiting and activation receptors (34) on NK cells control NK cell function in response to infection. Subsequently, NK cells engage in cytokine secretion and, upon the encounter of a virus-infected cell, release cytotoxic granule contents or induce apoptosis. These mechanisms lead to the elimination of virus-infected cells. Whereas the discovery of activating or inhibitory receptors on NK cells has progressed tremendously, the identification of respective ligands on infected or transformed cells has been difficult (reviewed in reference 12).Although much is known about the function of NK cells in humans and mice, NK cell activity in swine or cattle remains preliminary, and their role in animal viral diseases still is obscure. The recent progress in these animal species has been reviewed by Boysen and Storset (9) and Gerner et al. (20). In pigs, NK cells may account for a total of 5 to 10% of circulating lymphocytes and currently are identified as belonging to a subset of cells that coexpress CD2 and CD8 molecules (17). Although mRNAs of many activating and inhibitory receptors have been detected, no studies have been conducted to define their role in the generic function of porcine NK cells. But it is known that porcine NK cells can secret gamma interferon (IFN-γ), store perforin, and kill in vitro targets (54). Their function can be modulated by direct stimulation with cytokines such as interleukin-2 (IL-2), IL-12, IL-15, IL-18 (42, 54), or IFN-α, or Toll-like receptor (TLR) agonists such as polyinosinic:poly(C) (pI:C), CpG, imiquimod, and resiquimod in humans (23).In this study, we examine NK cell responses during infection with foot-and-mouth disease virus (FMDV). FMDV is a contagious disease of cloven-hoofed animals caused by a picornavirus (25). Infection with FMDV presents as an acute disease characterized by fever, short-lived viremia, and the occurrence of lesions on feet and tongue (reviewed in reference 25). The control of FMDV in certain regions of the world depends on the use of inactivated vaccines. The use of these as emergency vaccines, however, is compromised by the time from vaccination to protection (7 days in cattle) (22) and the difficulty in distinguishing infected animals from vaccinated animals. In FMDV-free countries vaccination is not practiced, leading to outbreak responses relying mostly on the elimination of all susceptible animals within the areas of outbreaks (15).The immune response to FMDV infection in pigs has not been fully dissected and remains an area of speculation. Whereas B-cell responses are activated early and neutralizing antibody titers correlate with protection (36), there is no clear picture regarding the induction of functional T-cell immunity in infected animals. Bautista et al. (4) found the inhibition of T-cell responses to mitogens during the infection of pigs with FMDV. Some groups have reported the induction of antigen-specific T cells reactive with nonstructural proteins of the virus (7). More recently, antigen-specific, major histocompatibility complex-restricted CD8⁺ T-cell responses were detected following infection with FMDV (26).The importance of innate immunity in FMDV infection is perhaps the most overlooked systemic response to FMDV in susceptible species and remains largely undefined. To date, there is no comprehensive study addressing the role of NK cells during infection with FMDV in either swine or cattle. A more sophisticated understanding of the innate response mechanisms involved in infection with FMDV is pertinent to the design of potent emergency vaccines that can protect against infection.In these studies, we characterized the response of NK cells derived from swine peripheral blood following infection with the O1 Campos strain of FMDV. Results demonstrate that these cells are compromised in the killing of either tumor cells or FMDV-infected target cells ex vivo. Moreover, this dysfunction includes a reduction in the proportion of IFN-γ-producing and perforin-storing cells and an alteration in the expression pattern of activating and inhibitory receptor genes. These data suggest a profound effect of FMDV infection on porcine NK cell function. Taken together, this abnormal functional status likely contributes to the acute nature of FMDV infection in this species and toward making it one of the most contagious viral infections of swine.     

Schistosoma mansoni egg antigen-mediated modulation of Toll-like receptor (TLR)-induced activation occurs independently of TLR2, TLR4, and MyD88

Unlike most pathogens, helminth parasites and their products induce strong Th2 responses, and dendritic cells (DCs) and macrophages exposed to helminth antigens generally fail to produce interleukin-12. Rather, it has been shown that helminth products such as soluble egg antigens (SEA; a soluble extract from Schistosoma mansoni eggs) inhibit the activation of DCs in response to classical Toll-like receptor (TLR) ligands such as lipopolysaccharide or CpG. Nevertheless, recent work has suggested that TLR4 and/or TLR2 plays an important role in the recognition of helminth products by DCs and macrophages and in the development of Th2 responses. Using DCs derived from TLR4^−/−, TLR2^−/−, or MyD88^−/− mice, we have demonstrated that the ability of SEA to modulate DC activation is MyD88 independent and requires neither TLR4 nor TLR2. Moreover, TLR2 and TLR4 are not required for SEA-pulsed DCs to induce Th2 responses in naïve mice.Helminth parasites, which colonize organ systems as diverse as the lymphatics, gastrointestinal tract, and vasculature, have evolved multiple immunomodulatory mechanisms to evade host immune responses (20). A delicate balance is required in these chronic infections to establish parasite survival without eliciting lethal immunopathology. This balance is illustrated in schistosomiasis, which is caused by the trematode Schistosoma mansoni and causes chronic morbidity in more than 200 million people (24). Following infection, worms migrate to the portal vasculature, where they mature and pair. This early phase of infection is characterized by a Th1 response. After worm pairing, females lay eggs that cross the intestinal barrier to be excreted in the feces. However, some eggs become lodged in the intestinal wall and liver sinusoids, where soluble egg antigens (SEA) induce a polarized Th2 response (23). The Th2 response correlates with the downmodulation of the initial proinflammatory Th1 response to migrating immature worms and results in granuloma formation. Failure to switch to a Th2 response leads to hepatotoxic liver disease and host death (3, 6, 11).The mechanism by which host innate immune cells recognize SEA remains unclear. Pathogens such as bacteria, viruses, and intracellular parasites express conserved molecular signatures that are shared within classes of pathogens and their free-living relatives. These are recognized by highly conserved pattern recognition receptors (PRRs) expressed by innate defense cells, including dendritic cells (DCs) and macrophages. PRRs include C-type lectins and Toll-like receptors (TLRs) (10, 32). TLRs are the most well-described PRRs, and DC activation by TLR ligation is considered to play a major role in the coordination of innate and adaptive immune responses during infection (22). Typically, the ligation of TLRs initiates a proinflammatory program, which promotes innate defense mechanisms and adaptive Th1 or Th17 immune responses to the invasive pathogens (22). There is evidence, however, that lipopolysaccharide (LPS) activation of TLR4 can induce DCs to support the development of Th2 responses (5, 15).Emerging data have demonstrated that phospholipids or glycoproteins unique to extracellular helminths ligate TLRs to induce an anti-inflammatory and Th2-inducing antigen-presenting cell phenotype. A phosphorylcholine-containing glycoprotein, ES-62, from the nematode Acanthocheilonema viteae, has been shown to induce a polarized Th2 response and to work via TLR4 to modulate antigen-presenting cell activation by a variety of TLR ligands (7, 33). There is also evidence that S. mansoni products can stimulate antigen-presenting cells through TLRs. A lipid fraction from S. mansoni eggs containing lysophosphatidylserine has been shown, in a TLR2-dependent mechanism, to induce the activation of DCs that promote Th2 and regulatory T-cell development (28), and lacto-N-fucopentaose III (LNFPIII), a synthetic copy of a schistosome egg glycan, has been shown to promote Th2 differentiation by DCs via a TLR4-dependent pathway (26).Here, using gene-targeted mice, we demonstrate conclusively that the anti-inflammatory and Th2-inducing characteristics of SEA are MyD88 independent and require neither TLR2 nor TLR4.     

Dendritic Cells in Uninfected Infants Born to Hepatitis B Virus-Positive Mothers

Plasmacytoid dendritic cells (pDCs) play a central role in antiviral immunity, detecting viruses via Toll-like receptors (TLR) and producing in response vast amounts of type I interferons (IFNs). Hepatitis B virus (HBV) causes chronic infection after vertical transmission. This study investigated whether an HBV-infected maternal environment might influence DC numbers and pDC function in uninfected infants. Blood was collected from inactive HBsAg carrier and control mothers and their infants at birth and 1 and 6 months of age. HBV DNA was measured in maternal and neonatal perinatal sera using real-time PCR. The circulating frequencies of myeloid DCs (mDCs) and pDCs were determined in the babies by flow cytometry. Peripheral blood mononuclear cells (PBMCs) and cord blood pDCs were stimulated with resiquimod, and alpha interferon (IFN-α) production and the pDC phenotype were assessed. The effect of the common-cold virus, rhinovirus (RV), on resiquimod stimulation was also determined. HBV DNA was detected in 62.3% of the mothers and 41% of their infants. DC numbers and pDC functions were similar between subjects and controls and were not correlated with maternal or neonatal viremia. RV infection did not induce pDC maturation until the age of 6 months, and it reduced TLR7-dependent resiquimod-induced IFN-α production similarly in both groups. Although the DC system is immature at birth, DCs of uninfected neonates of HBV-positive mothers are competent to initiate and maintain T-cell responses. RV is a weak inducer of IFN-α production until the age of 6 months and inhibits IFN-α responses triggered by the TLR7 pathway.Hepatitis B virus (HBV) is a hepatotropic noncytopathic DNA virus of the family Hepadnaviridae that causes a high rate (90%) of chronic infection when acquired through mother-to-infant transmission (16). The increased incidence of chronicity is attributed to the immaturity of the neonatal immune system and, specifically, to the functional impairment of T cells (1, 16). Neonatal dendritic cells (DCs) exhibit functional alterations that could lead to secondary defects of adaptive T-cell responses (2, 9, 12). The importance of DCs has been demonstrated by experiments showing that neonatal T cells can reach adult-like responses when stimulated with isolated allogeneic adult DCs (2). The main dysfunctions of neonatal DCs include low circulating numbers, low levels of costimulatory-molecule expression, decreased induction of cytokine production, and decreased capacity to stimulate naïve T cells (3, 12, 28).In humans, at least two distinct bone marrow-derived DC subsets have been characterized: those of myeloid (mDC) and of plasmacytoid (pDC) DC origin. In adults, DCs represent 0.8 to 1% of peripheral blood mononuclear cells (PBMCs) (5), whereas cord blood DCs (CB DCs) represent 0.3% of the CB mononuclear cells (CBMCs) (28). Upon exposure to pathogens, pDCs produce abundant amounts of type I/II interferons (IFNs), whereas mDCs produce high levels of interleukin 12 (IL-12). pDCs can produce 200 to 1,000 times more alpha interferon (IFN-α) than any other type of blood cell after they recognize viral genetic material through Toll-like receptors (TLRs) (11, 26). Thus, they represent a most important cell type in antiviral innate immunity. The favorable responses to IFN-α treatment in chronically infected HBV patients suggest that pDCs can play an important role in HBV infection. Indeed, several studies have found quantitative and qualitative impairment of pDCs in chronic carriers (5, 31).Although the mechanisms of mother-to-infant HBV transmission remain unclear, several factors have been shown to be involved, including high perinatal maternal viremia and transplacental passage of virions and viral antigens, as well as viral infection of neonatal PBMCs in both infected and uninfected infants (17, 18, 23, 30). It has been shown that exposure of PBMCs to HBV DNA in uninfected neonates can lead to defective T-cell responses and to HBV vaccination failure (30). Therefore, it can be speculated that even in the absence of neonatal infection, the presence of HBV or its products in the maternal environment may alter the development of the DC systems of these newborns. Similarly, in utero exposure to HIV-1 has been shown to induce quantitative and qualitative changes in uninfected neonatal DCs (27).Reports on the role of DCs in HBV infection have focused on adult life, after chronic infection has already been established (6). It is therefore important to study any alterations of the DC system during the neonatal period, when mother-to-infant HBV transmission may take place. The aim of the present study was to investigate whether the numbers and function of DCs may be altered in children of HBV-positive mothers compared with children born to healthy mothers. We measured the circulating frequencies of mDCs and pDCs and evaluated the capacity of pDCs to mature in response to resiquimod (R848), a well-known potent pDC activator. To understand if maternal viremia may influence the TLR7-dependent IFN-α-inducing pathway, we further assessed the effect of a common-cold virus, rhinovirus type 1b (RV1b), on TLR7 signaling post-R848 stimulation. RV is a single-stranded RNA (ssRNA) virus and hence a natural ligand of TLR7 (7, 14).     

Roles for NK Cells and an NK Cell-Independent Source of Intestinal Gamma Interferon for Innate Immunity to Cryptosporidium parvum Infection

A gamma interferon (IFN-γ)-dependent innate immune response operates against the intestinal parasite Cryptosporidium parvum in T- and B-cell-deficient SCID mice. Although NK cells are a major source of IFN-γ in innate immunity, their protective role against C. parvum has been unclear. The role of NK cells in innate immunity was investigated using Rag2^−/− mice, which lack T and B cells, and Rag2^−/− γ_c^−/− mice, which, in addition, lack NK cells. Adult mice of both knockout lines developed progressive chronic infections; however, on most days the level of oocyst excretion was higher in Rag2^−/− γ_c^−/− mice and these animals developed morbidity and died, whereas within the same period the Rag2^−/− mice appeared healthy. Neonatal mice of both mouse lines survived a rapid onset of infection that reached a higher intensity in Rag2^−/− γ_c^−/− mice. Significantly, similar levels of intestinal IFN-γ mRNA were expressed in Rag2^−/− and Rag2^−/− γ_c^−/− mice. Also, infections in each mouse line were exacerbated by treatment with anti-IFN-γ neutralizing antibodies. These results support a protective role for NK cells and IFN-γ in innate immunity against C. parvum. In addition, the study implies that an intestinal cell type other than NK cells may be an important source of IFN-γ during infection and that NK cells may have an IFN-γ-independent protective role.Cryptosporidiosis is an infectious diarrheal disease that affects different types of vertebrates, including mammals (3). The etiological agent is the monoxenous protozoan parasite Cryptosporidium, which belongs to the Apicomplexa. One species, Cryptosporidium hominis, may have a predilection for infecting humans, while a morphologically similar parasite, Cryptosporidium parvum, readily infects both cattle and humans (3). The cryptosporidia of mammals invade intestinal epithelial cells, where they multiply asexually to produce merozoites that infect more cells. Eventually, merozoites may undergo differentiation into gamonts that form new oocysts, containing four sporozoites, and the oocysts transmit infection to new hosts by the fecal-oral route. The clinical phase of cryptosporidiosis normally lasts a few days but may persist and become fatal in immunocompromised hosts (2).Studies of protective host immune responses to Cryptosporidium indicate that elimination of infection involves adaptive immunity and, in particular, requires the presence of CD4⁺ T cells. AIDS patients with low CD4⁺ cell counts have shown increased susceptibility to cryptosporidial infection and high rates of morbidity and mortality, while resolution of AIDS-associated infection following anti-human-immunodeficiency-virus drug treatment coincided with the partial recovery of intestinal CD4⁺ T-cell counts (2, 23). Mice with a CD4⁺ T-cell deficiency were found to be incapable of clearing C. parvum infection (1), and similarly, depletion of these cells from immunocompetent animals with specific antibody increased oocyst production (27). CD4⁺ T cells are also an important source of gamma interferon (IFN-γ), and this cytokine plays a key role in the control of infection. Antigen-specific IFN-γ production by restimulated CD4⁺ T cells from humans who recovered from infection was observed, although cells taken during acute infection were not responsive to antigen (6). IFN-γ^−/− mice or mice administered anti-IFN-γ neutralizing antibodies had exacerbated infections compared with control animals (18, 27). IFN-γ activity during C. parvum infection has been associated with a chemokine response by intestinal epithelial cells that attracted both CD4⁺ T cells and macrophages into the lamina propria (10). In addition, IFN-γ has been shown to have a direct effect on parasite growth by activating epithelial cell antimicrobial killing activity (19).Innate immune responses are also able to limit the reproduction of C. parvum. Immunocompromised adult nude mice (lacking T cells) or SCID mice (lacking T and B cells) developed chronic infections that were controlled for a number of weeks but eventually became progressive and fatal (13, 17, 27). IFN-γ was important for the initial resistance of these mice, since administration of anti-IFN-γ neutralizing antibodies to adult or neonatal SCID mice increased susceptibility to infection (14, 28), and repeated antibody treatment resulted in rapid establishment of severe infection (14). In addition, morbidity as a result of parasite reproduction appeared sooner in SCID IFN-γ^−/− mice than in SCID mice (7).NK cells are involved in resistance to intracellular microbial pathogens, including protozoa, and are a major source of IFN-γ in innate immunity (9). NK cells originate mainly in the bone marrow, from where they migrate to other organs (5, 29). Interleukin-15 (IL-15) is essential for differentiation and subsequent survival of NK cells and can also be important in activation of the cells (5, 9). NK cells are activated by ancillary cells, such as dendritic cells (DCs), by direct contact and by proinflammatory cytokines produced by DCs stimulated by antigen (9). Activated NK cells produce IFN-γ and other proinflammatory cytokines and may also become cytotoxic against infected cells.The protective role of NK cells in innate immunity to C. parvum is unclear, but some studies imply that these cells may be involved. Human peripheral blood NK cells treated with IL-15 were shown to have cytolytic activity against human intestinal epithelial cell lines infected with C. parvum (4), and intestinal expression of this cytokine has been detected in humans (20). C. parvum infection was found to be more widespread in SCID mice deficient in NK cell cytotoxicity than in SCID mice with normal NK cell function (17). In addition, in vitro studies demonstrated that splenocytes from SCID mice produced IFN-γ in the presence of cryptosporidial antigens, but if NK cells were depleted, IFN-γ production did not occur (15). However, attempts to show that NK cells were protective in SCID mice infected with C. parvum have not been successful. In separate studies, treatment of these mice with anti-asialo-GM1 antibodies that can deplete NK cells in vivo was shown to have no effect on the course of C. parvum infection (15, 27), and while it has been argued that these antibodies might not have reached the gut in sufficient quantity to be effective, similar antibodies were shown to diminish intestinal NK cell function (30).The aim of the present study was to examine further the role of NK cells and IFN-γ in the innate immune response to C. parvum. The pattern of infection and immune responses were compared in Rag2^−/− mice, which lack T and B cells, and Rag2^−/− γ_c^−/− mice, which, in addition, lack NK cells due to the absence of the γ_c chain component of the IL-15 receptor (5). The results support protective roles for IFN-γ and NK cells in innate immunity to C. parvum but also indicate that IFN-γ from a cell type other than NK cells is important for control of infection.     

Transitions in Oral and Intestinal Microflora Composition and Innate Immune Receptor-Dependent Stimulation during Mouse Development

Commensal bacteria possess immunostimulatory activities that can modulate host responses to affect development and homeostasis in the intestine. However, how different populations of resident bacteria stimulate the immune system remains largely unknown. We characterized here the ability of intestinal and oral microflora to stimulate individual pattern recognition receptors (PRRs) in bone marrow-derived macrophages and mesothelial cells. The intestinal but not oral microflora elicited age- and cell type-specific immunostimulation. The immunostimulatory activity of the intestinal microflora varied among individual mice but was largely mediated via Toll-like receptor 4 (TLR4) during breast-feeding, whereas it became TLR4 independent after weaning. This transition was associated with a change from a microflora rich in TLR4-stimulatory proteobacteria to one dominated by Bacteroidales and/or Clostridiales that poorly stimulate TLR4. The major stimulatory activity of the intestinal microflora was still intact in NOD1-, NOD2-, TLR2-, TLR4-, TLR5-, TLR9-, TLR11-, ASC-, or RICK-deficient cells but still relied on the adaptor MyD88. These studies demonstrate a transition in the intestinal microflora accompanied by a dynamic change of its ability to stimulate different PRRs which control intestinal homeostasis.Accumulating evidence indicates that environmental bacteria can regulate the development and homeostasis of the host immune system, particularly within the gut, and affect susceptibility to a variety of diseases (3, 5, 6, 9, 10, 40, 44). Both humans and animals harbor a large number of nonpathogenic residential bacteria, especially in the intestine and oral cavity (41). Uncontrolled translocation of bacteria or bacterial components into systemic tissues of the host often results in bacteremia and sepsis (8), which causes significant mortality worldwide each year. On the other hand, intestinal bacteria contain immunostimulatory molecules that can regulate local immunity, epithelial development, immunotolerance, and susceptibility to inflammatory bowel disease (2, 41). The bacterium-derived molecules are recognized by innate immune receptors, including Toll-like receptors (TLRs) and Nod-like receptors (NLRs) (8, 20). TLRs and NLRs, often referred as pattern recognition receptors (PRRs), are involved in the recognition of commensal and pathogenic bacteria, as well as in the clearance of pathogens through interaction with their cognate microbial molecules (8, 20). Interactions between PRRs and commensal bacteria have been demonstrated to be important for gut homeostasis. MyD88, an essential adaptor for TLR signaling, has been shown to be important for epithelial homeostasis (40) and IgA secretion in the intestine (3, 44), and TLR9 has been shown to be important for the balance of regulatory T/Th17/Th1 cells (10). In addition, NOD1, an NLR family member, was shown to play a role in the development of intestinal lymphoid tissue via the recognition of commensal bacteria (5). Finally, genetic variation in NOD1 affects the susceptibility to allergic disease (17, 49) and Crohn''s disease (31) and that in NOD2 regulates susceptibility to Crohn''s disease (11, 16, 36) and graft-versus-host disease (14). In the oral cavity, the immunostimulatory properties of periodontal bacteria are believed to be important for the development of dental diseases (47).Although beneficial interactions between commensal bacteria and innate immune receptors have been demonstrated, the dominant bacterial species that are responsible for PRR stimulation in the normal intestine and oral cavity are unknown. Moreover, the commensal-dependent immunostimulatory activity for the various innate immune receptors has not been characterized. In humans, both genetic factors and the environment, including the diet, modulate the composition of the microflora (7, 15, 23, 33, 41, 48). Consequently, the intestinal microflora of individual humans is highly diverse (7, 15, 23, 33, 41). By taking advantage of the mouse model which allows a more uniform environmental and genetic platform, we analyzed the immunostimulatory activity induced by the oral and intestinal microflora and linked the activity to specific populations of commensal bacteria that emerge during postnatal development. We study provide here a comprehensive resource on microflora analysis in the mouse that is expected to facilitate future studies of the interaction between commensal bacteria and host immunity.     

Cooperation between Multiple Microbial Pattern Recognition Systems Is Important for Host Protection against the Intracellular Pathogen Legionella pneumophila

Multiple pattern recognition systems have been shown to initiate innate immune responses to microbial pathogens. The degree to which these detection systems cooperate with each other to provide host protection is unknown. Here, we investigated the importance of several immune surveillance pathways in protecting mice against lethal infection by the intracellular pathogen Legionella pneumophila, the causative agent of a severe pneumonia called Legionnaires'' disease. Rip2 and Naip5/NLRC4 signaling was found to contribute to the innate immune response generated against L. pneumophila in the lung. Elimination of Rip2 or Naip5/NLRC4 signaling in MyD88-deficient mice resulted in increased replication and dissemination of L. pneumophila and higher rates of mortality. Irradiated wild-type mice receiving bone marrow cells from pattern recognition receptor-deficient mice displayed L. pneumophila infection phenotypes similar to those of donor mice. Rip2 and Naip5/NLRC4 signaling provided additive effects in protecting MyD88-deficient mice from lethal infection by L. pneumophila, with the contribution of Naip5/NLRC4 being slightly greater than that of Rip2. Thus, activation of the Rip2, MyD88, and Naip5/NLRC4 signaling pathways triggers a coordinated and synergistic response that protects the host against lethal infection by L. pneumophila. These data provide new insight into how different pattern recognition systems interact functionally to generate innate immune responses that protect the host from lethal infection by activating cellular pathways that restrict intracellular replication of L. pneumophila and by recruiting to the site of infection additional phagocytes that eliminate extracellular bacteria.To respond to diverse populations of microbes, the mammalian innate immune system utilizes germ line-encoded pattern recognition receptors (PRRs) that detect conserved molecular patterns associated with pathogens (38). The ectodomains of transmembrane Toll-like receptor (TLR) are involved in detecting microbes outside cells and within vacuoles, and the adapter protein MyD88 is used by many TLRs to transduce extracellular signals into functional responses (38). In contrast, the nucleotide-binding domain, leucine-rich repeat (NLR) proteins constitute a surveillance mechanism capable of responding to microbial products delivered into the host cytosol (27). The Nod1 and Nod2 proteins are PRRs that detect microbial products present in the cytosol and in response activate NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways through an adapter serine-threonine kinase called Rip2 (11, 18, 25, 26, 28, 29, 33, 44, 46, 50).The Gram-negative bacterium Legionella pneumophila is a useful model for investigating the initiation of the innate immune response. L. pneumophila persists in the environment as a parasite of freshwater protozoans (15); however, upon gaining access to the mammalian respiratory system through contaminated aerosols, the bacteria can infect and replicate within alveolar macrophages (17, 24, 37). Failure to treat infected individuals, especially those who are immunocompromised, with antibiotics can lead to the development of a severe pneumonia known as Legionnaires'' disease (17, 37). Following phagocytosis by a macrophage, L. pneumophila generates a unique vacuole that evades fusion with lysosomes and accumulates endoplasmic reticulum (ER) protein markers, features that allow the compartment to support intracellular replication (12, 22, 23, 30, 56). L. pneumophila is able to perform this task by utilizing a type IV secretion system encoded by the dot and icm genes (36, 48, 57). The Dot/Icm secretion apparatus delivers bacterial proteins into the host cell cytosol that modulate normal endosomal trafficking and prevent lysosome-mediated killing of the bacteria (31, 41).The proteins TLR2, TLR5, and TLR9 have been shown to recognize L. pneumophila during engulfment at the cell surface or in an early endosomal compartment (2, 6, 7, 19-21, 43). Mice deficient in TLR2 have a subtle defect in clearance of L. pneumophila from the lung after infection (6, 20). Surprisingly, defects in TLR5 and TLR9 signaling do not exacerbate this TLR2 defect significantly (5), suggesting that TLR signaling alone is not essential for host protection against L. pneumophila infection. Mice deficient for MyD88 have a profound defect in interleukin-12 (IL-12) and gamma interferon (IFN-γ) production (5, 6, 20, 54) and display high numbers of L. pneumophila CFU in the lungs compared to control mice (6, 20). MyD88 is required for signaling pathways stimulated by TLRs and for pathways activated by the IL-1 family of receptors (1), which is the likely reason why a deficiency in MyD88 results in a more severe L. pneumophila susceptibility phenotype than a deficiency in the three primary TLRs stimulated by L. pneumophila. Macrophages and NK cells have been implicated as cell types that utilize MyD88 for an in vivo response to L. pneumophila (5, 6, 20, 54); however, it remains to be determined which cell types play a protective role in the MyD88-dependent response.In addition to activating MyD88-dependent pathways, virulent L. pneumophila activates cytosolic pattern recognition systems. The flagellin protein produced by L. pneumophila signals through the NLR proteins Naip5 and NLRC4 (also known as IPAF and CARD12), resulting in the activation of caspase-1 and other pathways that restrict intracellular replication of L. pneumophila in mouse macrophages (4, 34, 40, 45, 58). Increased replication of L. pneumophila in the lungs is observed after infection of mice deficient in Naip5 or NLRC4 signaling (4, 10, 34, 58); however, these mice are still able to clear the infection over a period of several days. The finding that L. pneumophila activates a Rip2-dependent signaling pathway in macrophages that mediates IκB degradation and NF-κB nuclear translocation suggests that the NLR proteins Nod1 and Nod2 are also involved in detection (35, 52). Whether Rip2 signaling is important for host protection against L. pneumophila, however, has not been addressed.The ability of multiple pathogen recognition systems to respond to L. pneumophila makes this an attractive model to investigate whether these different signaling pathways play functionally independent or synergistic roles in stimulating the host defense to this intracellular pathogen. In this study, we used a mouse model of Legionnaires'' disease to investigate the role of multiple microbial recognition systems in providing host protection against this intracellular pathogen.     

Streptococcus pneumoniae Autolysis Prevents Phagocytosis and Production of Phagocyte-Activating Cytokines

Streptococcus pneumoniae is a major pathogen in humans. The pathogenicity of this organism is related to its many virulence factors, the most important of which is the thick pneumococcal capsule that minimizes phagocytosis. Another virulence-associated trait is the tendency of this bacterium to undergo autolysis in stationary phase through activation of the cell wall-bound amidase LytA, which breaks down peptidoglycan. The exact function of autolysis in pneumococcal pathogenesis is, however, unclear. Here, we show the selective and specific inefficiency of wild-type S. pneumoniae for inducing production of phagocyte-activating cytokines in human peripheral blood mononuclear cells (PBMC). Indeed, clinical pneumococcal strains induced production of 30-fold less tumor necrosis factor (TNF), 15-fold less gamma interferon (IFN-γ), and only negligible amounts of interleukin-12 (IL-12) compared with other closely related Streptococcus species, whereas the levels of induction of IL-6, IL-8, and IL-10 production were similar. If pneumococcal LytA was inactivated by mutation or by culture in a medium containing excess choline, the pneumococci induced production of significantly more TNF, IFN-γ, and IL-12 in PBMC, whereas the production of IL-6, IL-8, and IL-10 was unaffected. Further, adding autolyzed pneumococci to intact bacteria inhibited production of TNF, IFN-γ, and IL-12 in a dose-dependent manner but did not inhibit production of IL-6, IL-8, and IL-10 in response to the intact bacteria. Fragments from autolyzed bacteria inhibited phagocytosis of intact bacteria and reduced the in vitro elimination of pneumococci from human blood. Our results suggest that fragments generated by autolysis of bacteria with reduced viability interfere with phagocyte-mediated elimination of live pneumococci.The pneumococcus Streptococcus pneumoniae is a leading cause of community-acquired pneumonia, meningitis, otitis media, and sinusitis and is a common cause of infection-related mortality in children and elderly people (28, 37).There is a large number of streptococcal species whose taxonomic classification is debated (14, 31). A number of streptococci, including alpha-hemolytic and nonhemolytic variants, constitute the viridans group, which can be further subdivided into the mitis, sanguinis, anginosus, salivarius, and mutans groups based on biochemical tests (14). Phenotypic and genetic tests consistently show that S. pneumoniae is closely related to and may be placed in the mitis subgroup (14, 30). Although the other members of the mitis group can cause sepsis and endocarditis (53), they are considerably less virulent than S. pneumoniae.Pneumococci are considered strictly extracellular pathogens, whose elimination depends on ingestion and killing by phagocytes (i.e., alveolar and tissue-resident macrophages and neutrophils recruited during the inflammatory process). Accordingly, an important determinant of pneumococcal pathogenicity is the thick, hydrophilic polysaccharide capsule, which impedes elimination by phagocytes in the absence of capsule-specific antibodies.The ability of phagocytes to kill microbes is augmented by the phagocyte-activating cytokines gamma interferon (IFN-γ) and tumor necrosis factor (TNF), which boost the bactericidal machinery and enhance killing and digestion of bacteria present within the phagosome (4, 39, 47). TNF is produced by monocytes/macrophages and activated T cells, while IFN-γ is produced by NK cells and T cells in response to interleukin-12 (IL-12) from macrophages. Thus, production of TNF, IFN-γ, and IL-12 is necessary for host defense against intracellular bacteria (8, 11, 21, 34, 48). More recently, these phagocyte-activating cytokines have also been shown to be essential for controlling extracellular gram-positive bacteria, including S. pneumoniae (36, 42, 50, 52, 54). Thus, a patient with an IL-12 deficiency was shown to suffer from recurrent episodes of pneumococcal infection (20). Phagocyte activation by TNF and/or IFN-γ might be required for decomposition of the thick, sturdy peptidoglycan (PG) layer of gram-positive bacteria after phagocytosis, while gram-negative bacteria may be more easily digested. Thus, human leukocytes produce more TNF, IFN-γ, and IL-12 when they are stimulated with gram-positive bacteria than when they are stimulated with gram-negative bacteria (23, 24).A peculiar property of S. pneumoniae is its tendency to undergo autolysis when it reaches the stationary phase of growth. This process is mediated by enzymes called autolysins (ALs), which, when activated, degrade cell wall PG. The major AL is an N-acetyl-muramyl-l-alanine amidase called LytA (27). Other pneumococcal ALs include LytB and LytC, which are believed to be involved mainly in modification of the cell wall during growth and division (16, 17). ALs are anchored to the cell wall via interactions with choline moieties on teichoic acid and lipoteichoic acid (LTA). Choline is necessary for pneumococcal growth, but culture in the presence of high concentrations of choline renders the bacteria incapable of undergoing autolysis (6, 19).Studies with mice have shown that S. pneumoniae with mutated LytA is less virulent than wild-type pneumococci (2, 7, 25). The reason for this is not clear, but two main hypotheses have been put forward. First, autolysis promotes the release of the intracellular toxin pneumolysin (Ply) (5, 33). Ply is an important determinant of virulence (3, 41) and interferes with several defense systems, including inhibition of ciliary beating (15), complement activation (38), and induction of intracellular oxygen radical production (33). Second, cell wall degradation products, such as soluble PG fragments and LTA released upon autolysis, have been suggested to augment the inflammatory response (9, 10, 44, 49).Here we examine a third possibility, that autolysis interferes with the generation of phagocyte-activating cytokines. We have previously shown that intact gram-positive bacteria provide a very efficient stimulus for IL-12 production by human monocytes, regardless of whether they are dead or alive (1, 23, 24), but that decomposed bacteria are inactive in this process and soluble components of the gram-positive cell wall, such as PG and LTA, even downregulate the production of IL-12 in response to intact bacteria in a dose-dependent manner (1). These observations led us to speculate that autolysis may promote virulence by generating bacterial cell wall fragments that block IL-12 production and thereby reduce IFN-γ production and phagocyte activation. Indeed, our data demonstrate that AL-mediated disintegration of pneumococci inhibits production of IFN-γ and also TNF in response to intact bacteria. Further, the presence of autolyzed bacteria reduced elimination of live pneumococci by blood cells in vitro.     

Human Brucellosis Is Characterized by an Intense Th1 Profile Associated with a Defective Monocyte Function

In animal models, a defective Th1 response appears to be critical in the pathogenesis of brucellosis, but the Th1 response in human brucellosis patients remains partially undefined. Peripheral blood from 24 brucellosis patients was studied before and 45 days after antibiotherapy. Twenty-four sex- and age-matched healthy donors were analyzed in parallel. Significantly increased levels of interleukin 1β (IL-1β), IL-2, IL-4, IL-6, IL-12p40, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), but not of IL-10, in serum and/or significantly increased percentages of samples with detectable levels of these cytokines, measured by enzyme-linked immunosorbent assays (ELISA), were found for untreated brucellosis patients, but these levels were reduced and/or normalized after treatment. Flow cytometry studies showed that the intracytoplasmic expression of IFN-γ, IL-2, and TNF-α, but not that of IL-4, by phorbol myristate-activated CD4⁺ CD3⁺ and CD8⁺ CD3⁺ T lymphocytes was significantly increased in untreated brucellosis patients and was also partially normalized after antibiotherapy. The percentage of phagocytic cells, the mean phagocytic activity per cell, and the phagocytic indices for monocytes at baseline were defective and had only partially reverted at follow-up. T lymphocytes from untreated brucellosis patients are activated in vivo and show Th1 cytokine production polarization, with strikingly high serum IFN-γ levels. In spite of this Th1 environment, we found deficient effector phagocytic activity in peripheral blood monocytes.Brucellosis is a zoonotic disease of worldwide distribution. Despite its control in many countries, it remains endemic in the Mediterranean and Middle Eastern regions (20, 28, 41, 42). Brucella melitensis is the most frequent cause of human brucellosis in these geographical areas (19). In Spain, it has been reported that the majority (more than 97.5%) of isolates were identified as Brucella melitensis (13, 44, 45).Brucella organisms are facultatively intracellular Gram-negative coccobacilli that reside and replicate in a vacuolar compartment within myelomonocytic cells of the infected host (14, 15, 47). The response to Brucella involves the whole gamut of the immune system, from innate to adaptive immunity (21). In murine models, passive transfer of immune cells resulted in an effective anti-Brucella defensive response mediated by CD4⁺ and CD8⁺ T lymphocytes (5, 6, 32, 37, 51, 52). Furthermore, the pattern of T-lymphocyte cytokine secretion is considered to be critical for the effectiveness of the protective anti-Brucella immune response (3, 7). It has been postulated that Th1 cytokines confer resistance, while Th2 cytokines facilitate the development of brucellosis (2, 3, 24, 25, 40, 43, 52). In animal models, gamma interferon (IFN-γ) induces macrophage activation and control of Brucella infection (16, 18, 43). In Brucella-infected mice, administration of recombinant IFN-γ enhances host resistance, resulting in a deep decrease in the number of viable bacteria (51). Moreover, host IFN-γ depletion results in an increase in the number of viable bacteria (17, 37, 52). Several abnormalities in the immune system have been found in human brucellosis (27, 46, 49). It has been found that T and NK lymphocytes show defective functions in brucellosis patients (46, 49). Since mice are naturally resistant to Brucella infections, it is possible to suggest that the immune response elicited by Brucella in humans might have different characteristics. Thus, susceptibility to, or protection from, human brucellosis conferred by T-lymphocyte cytokines has not been established.In this work, we have further investigated the pattern of T-lymphocyte and monocyte responses to human Brucella infection. We have prospectively studied (i) the levels of Th1, Th2, and regulatory cytokines in serum, (ii) the distribution, activation stage, and pattern of Th1/Th2 cytokine production by T lymphocytes, and (iii) the phagocytic activity of monocytes in a group of brucellosis patients before and after antimicrobial treatment.     

Toll-Like Receptor 3 Ligand and Retinoic Acid Enhance Germinal Center Formation and Increase the Tetanus Toxoid Vaccine Response

Immunizations with T-cell-dependent antigens induce the formation of germinal centers (GC), unique lymphoid microenvironments in which antigen-activated B cells undergo class switching, affinity maturation, and differentiation into memory B cells. Poly(I:C) (PIC), a double-stranded RNA, and retinoic acid (RA), a metabolite of vitamin A which induces cell differentiation, have been shown to augment both primary and memory anti-tetanus toxoid (anti-TT) IgG responses. However, their influence on the GC reaction is unknown. In the present study, 6-week-old C57BL/6 mice were immunized with TT and cotreated with PIC, RA, or both. The splenic GC reaction was evaluated using immunofluorescence staining 10 days after TT priming. Each treatment enhanced the TT-induced GC formation (number of GC/follicle and GC area) about two- to threefold, which correlated with the titers of plasma anti-TT immunoglobulin G (IgG). Isotype switching to IgG1 was dramatically stimulated, with the greatest increase in IgG1-positive GC B cells induced by RA-PIC (P < 0.001). Moreover, PIC alone and RA-PIC robustly promoted the formation of the follicular dendritic cell (FDC) network in the GC light zone. PIC and RA-PIC also increased IgG1-positive B cells in the periarterial lymphatic sheath regions, where most IgG1-positive cells were plasma cells (CD138/syndecan-1 positive), suggesting that plasma cell generation was also enhanced in non-GC regions. The stimulation of several processes, including antigen-induced GC formation, isotype switching, FDC network formation within GC, and plasma cell differentiation by RA and/or PIC, suggests that this nutritional-immunological combination could be an effective means of promoting a robust vaccine response.Successful vaccination with protein antigens depends on the differentiation and maturation of antigen-activated B cells into class-switched, long-lived memory B cells. The germinal center (GC) reaction is crucial to these processes. Upon infection or immunization, some antigen-activated B cells that have encountered cognate antigen-activated T helper (Th) cells differentiate directly into antibody-secreting plasma cells and produce antibodies that provide a level of initial protection. However, other antigen-activated B cells migrate into the secondary follicles of lymphoid organs, including spleen and lymph nodes, and participate in the GC reaction, which comprises the formation a dynamic antigen-induced microenvironment in which B cells can differentiate and mature, undergoing processes of class switch recombination and somatic hypermutation (12, 40, 52, 54). Typically, GCs exhibit polarization into two zonal regions, termed the dark zone and the light zone. In the dark zone, newly stimulated B cells proliferate rapidly and undergo somatic mutation. The progeny of these B cells, centrocytes, migrate into the light zone, where follicular dendritic cells (FDC) (1), together with antigen-activated Th cells, provide essential signals for B-cell survival, class switch recombination, affinity maturation, and differentiation into long-lived plasma cells or memory B cells (5, 10, 32, 54). Hence, the formation of the GC structure and the cellular and molecular processes that occur within GCs are essential for the generation of B cells expressing antibodies of the immunoglobulin G (IgG), IgA, or IgE class, with high-affinity antigen-combining sites, as well as for the production of memory B cells that, on reactivation, will produce a rapid, high-output response capable of neutralizing the pathogen and protecting the infected host from developing life-threatening disease (32).The Toll-like receptor (TLR) family is a group of pattern recognition receptors that are mostly distributed on monocytes and dendritic cells (7, 56). Various TLR members bind conserved microbial structures, initiating reactions that are essential for innate immunity and immunoregulatory for adaptive immunity (39). Thus, TLR ligands are now considered important adjuvant targets for vaccine development (21, 45). Poly(I:C) (PIC), a synthetic double-stranded polyribonucleotide composed of polyriboinosinic acid paired with polyribocytidylic acid, is a mimetic of double-stranded RNA viruses (59). PIC binds to TLR3/MDA5 and activates downstream signaling pathways (21). TLR3 is especially implicated in sensing viral infections and in activating innate immunity (31, 58). PIC has long been known for its ability to induce type I and type II interferons (25), stimulate the cytotoxic activity of NK cells (4, 41, 59), and increase antiviral and antitumor reactions (26, 50, 51). Moreover, PIC is a potent activator of adaptive immunity through induction of dendritic cell maturation, regulation of Th1/Th2 responses, and promotion of antigen-specific antibody responses (4, 59), and TLR ligands have been suggested to promote GC formation (42).Retinoic acid (RA), an active metabolite of vitamin A (retinol), is well known as a potent agent of cell differentiation, including for B cells (9, 11, 18, 33, 36) and other immunoregulatory cells. Vitamin A, RA, and related retinoids have been shown to be necessary for maintaining antigen-specific antibody responses (47). Vitamin A deficiency causes low and dysregulated primary and memory antibody responses against several T-cell-dependent (TD) and polysaccharide antigens (47, 57), including common vaccines, such as tetanus and diphtheria toxoids (46). These impairments are reversible, since providing vitamin A or RA to vitamin A-deficient animals resulted in significant rescue of the anti-tetanus antibody response (16, 24). In humans, supplementation with vitamin A is recognized as an effective means of reducing morbidity and mortality among young children, a population at risk for vitamin A deficiency (53), and vitamin A supplementation has shown therapeutic benefits in infectious diseases, such as measles and diarrhea (53, 57, 60). In animals with normal vitamin A status, RA can act as a potent immunological adjuvant. Recently, we have reviewed studies showing that RA augments the production of an antigen-specific B-cell response in vivo (48). RA has gained attention due to its multiple effects on innate and adaptive immunity, including its ability to modulate cytokine production (27), promote the development of Th2 cells (20), induce gut-homing T cells (22) and T regulatory cells (6), regulate Th17 cells (37), stimulate B-cell maturation (11, 62), and increase primary and memory antibody responses (14, 16, 27). The potentiation of TD antigen-specific antibody production after treatment with RA could be due to a direct involvement in B-cell activation, as RA enhanced the production of IgM and IgG induced by both TD and T-cell-independent antigen in human peripheral blood mononuclear cells (61) and stimulated the plasmacytic maturation of isolated B cells in vitro (11). RA also elevated IgG and IgG1 production triggered by anti-μ or anti-CD40 in vitro (11, 36), stimulated gut-homing B cells (35), and increased the activation of the B-cell memory pool present in human peripheral blood lymphocytes (18). Thus, retinoids may have pleiotropic effects on the development of immune responses after infection and vaccination. However, to our knowledge, the ability of retinoids to modulate GC formation has not been investigated.On the basis of our previous reports that the combination of RA and PIC, when administered to normal adult or neonatal mice at the time of tetanus toxoid (TT) priming, strongly increased the primary anti-TT IgG response, the number of anti-TT IgG-secreting plasma cells (27, 28), and the anti-TT IgG memory response elicited at a later time by TT alone (27, 28), we hypothesized that PIC and RA influence the initial antigen-induced GC reaction. Therefore, the present study was designed to assess immunocytochemical changes in the spleen and their correlation with plasma antibody titers to test whether PIC and RA, given either alone or together, promote the GC response to TT, a clinically important TD antigen.     

Toll-Like Receptor Stimulation Enhances Phagocytosis and Intracellular Killing of Nonencapsulated and Encapsulated Streptococcus pneumoniae by Murine Microglia

Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity that are expressed in microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the responses against Streptococcus pneumoniae, the most common agent causing bacterial meningitis beyond the neonatal period. Murine microglial cultures were stimulated with agonists for TLR1/2 (Pam₃CSK₄), TLR4 (lipopolysaccharide), and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed to either the encapsulated D39 (serotype 2) or the nonencapsulated R6 strain of S. pneumoniae. After stimulation, the levels of interleukin-6 and CCL5 (RANTES [regulated upon activation normal T-cell expressed and secreted]) were increased, confirming microglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteria than unstimulated cells (P < 0.05). The presence of cytochalasin D, an inhibitor of actin polymerizaton, blocked >90% of phagocytosis. Along with an increased phagocytic activity, the intracellular bacterial killing was also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest that microglial stimulation by these TLRs may increase the resistance of the brain against pneumococcal infections.Immunocompromised patients have a higher risk of developing bacterial infections in the central nervous system (CNS) (34, 37, 42). The list of the pathogens includes many organisms with low pathogenicity in the immunocompetent host (34, 37). Moreover, the distribution of the pathogens also differs from the immunocompetent host and depends on the nature of the immune defect. Patients with a decrease in B-lymphocyte function or with a loss of splenic function have an increased risk of meningitis caused by encapsulated bacteria, while patients with an impaired T-lymphocyte-macrophage system are more susceptible to CNS infections caused by intracellular pathogens (7, 42). One additional cause of this increased susceptibility to CNS infections probably is a decreased local immune defense (33).CNS infections not only are more frequent but also are associated with higher mortality rates and more severe long-term sequelae in immunocompromised than in immunocompetent individuals (9, 17, 34, 44). Polymicrobial infections, multiple organ system presentation, and the absence of typical clinical manifestations subsequent to the host''s diminished inflammatory response are challenging aspects in the management of these infections (34, 37, 42).The brain tissue shows a well-organized innate immune reaction in response to bacteria in the cerebrospinal fluid (CSF) (3, 21). Microglial cells, the resident phagocytes of the CNS, express Toll-like receptors (TLRs) that identify pathogen-associated molecular patterns (PAMPs) (41). The receptor-ligand interactions activate microglia to undergo morphological transformation as well as functional changes, such as the production of proinflammatory cytokines, chemokines, and reactive oxygen species, enhanced phagocytic activity, and antigen presentation (15, 39). This immune reaction cannot eliminate high amounts of pneumococci from the CSF but does prevent or minimize the invasion of these pathogens into the brain tissue, thereby limiting tissue destruction and neuronal injury.TLR2, -4, and -9 contribute to the recognition and response to Streptococcus pneumoniae in the CNS (31). A deficiency of TLR2, -4, or -9 or of the coreceptor CD14, which is necessary for TLR4 signaling increases the susceptibility of mice to S. pneumoniae (1, 11, 12, 40).Here, we hypothesized that activation of the innate immune response in microglia could increase the resistance of the brain tissue against CNS pneumococcal infections (14). This may be of particular interest in immunocompromised patients, whose outcome after S. pneumoniae meningitis is worse than that of immunocompetent individuals (9, 44). The aim of the present study was to investigate whether the stimulation of microglia by respective PAMPs can increase their ability to phagocytose and kill intracellular nonencapsulated and encapsulated S. pneumoniae strains, thereby protecting the brain during meningitis. Moreover, by using an encapsulated and a nonencapsulated pneumococcal strain, we assessed the protective effect of the capsule against phagocytosis by microglial cells.