α-Galactosylceramide Promotes Killing of Listeria monocytogenes within the Macrophage Phagosome through Invariant NKT-Cell Activation

α-Galactosylceramide (α-GalCer) has been exploited for the treatment of microbial infections. Although amelioration of infection by α-GalCer involves invariant natural killer T (iNKT)-cell activation, it remains to be determined whether macrophages (Mφ) participate in the control of microbial pathogens. In the present study, we examined the participation of Mφ in immune intervention in infection by α-GalCer using a murine model of listeriosis. Phagocytic and bactericidal activities of peritoneal Mφ from C57BL/6 mice, but not iNKT cell-deficient mice, were enhanced after intraperitoneal injection of α-GalCer despite the absence of iNKT cells in the peritoneal cavity. High levels of gamma interferon (IFN-γ) and nitric oxide (NO) were detected in the peritoneal cavities of mice treated with α-GalCer and in culture supernatants of peritoneal Mφ from mice treated with α-GalCer, respectively. Although enhanced bactericidal activity of peritoneal Mφ by α-GalCer was abrogated by endogenous IFN-γ neutralization, this was only marginally affected by NO inhibition. Similar results were obtained by using a listeriolysin O-deficient strain of Listeria monocytogenes. Moreover, respiratory burst in Mφ was increased after α-GalCer treatment. Our results suggest that amelioration of listeriosis by α-GalCer is, in part, caused by enhanced killing of L. monocytogenes within phagosomes of Mφ activated by IFN-γ from iNKT cells residing in an organ(s) other than the peritoneal cavity.Listeria monocytogenes, a Gram-positive facultative intracellular bacterium, is the causative agent of listeriosis, with an overall mortality rate of 30% (76). A major virulence factor of L. monocytogenes is listeriolysin O (LLO), a 58-kDa protein encoded by the hly gene (26, 42, 65). LLO promotes intracellular survival of L. monocytogenes in professional phagocytes such as macrophages (Mφ) by promoting listerial escape from the phagosome into the cytosol (10, 22, 26, 42, 62, 65). Cells of the innate immune system play a pivotal role as a first line of defense against L. monocytogenes infection and among these, mononuclear phagocytes are critical (56, 61). Activation of Mφ by gamma interferon (IFN-γ) is mandatory for elimination of L. monocytogenes (31, 35). Nitric oxide (NO) synthesized by inducible NO synthase, which is localized in the cytosol of professional phagocytes, participates in killing of L. monocytogenes (48, 52, 69, 71). Similarly, reactive oxygen intermediates (ROI) play a role in killing of L. monocytogenes within the phagosome (52, 53, 59).Natural killer T (NKT) cells represent a unique T-lymphocyte population expressing NKR-P1B/C (NK1.1; CD161), which is a type 2 membrane glycoprotein of the C-type lectin superfamily (6). In the mouse, the majority of NKT cells express an invariant T-cell receptor (TCR) α chain encoded by Vα14/Jα18 gene segments and a TCRVβ highly biased toward Vβ8.2, Vβ7, and Vβ2 (invariant NKT [iNKT] cells) (6). In contrast to conventional T cells, which recognize antigenic peptides presented by polymorphic major histocompatibility complex class I or class II molecules, iNKT cells recognize glycolipid antigens, including α-galactosylceramide (α-GalCer), a synthetic glycolipid originally isolated from a marine sponge, presented by the nonpolymorphic antigen presentation molecule CD1d (6, 40). iNKT cells are highly versatile and promptly produce both type 1 and type 2 cytokines, such as IFN-γ and interleukin-4 (IL-4), respectively, upon activation through their TCRs (1, 15-17, 79). IL-15 is an essential growth factor of both iNKT cells and NK cells and, hence, both cell populations are absent in IL-15-deficient (IL-15^−/−) mice (58). The numbers of iNKT cells are also markedly reduced in SJL mice because of a large deletion in their TCRVβ genetic region (5, 78).In vivo administration of α-GalCer causes prompt release of various cytokines by iNKT cells, which are involved in the control of various diseases, e.g., tumor rejection and prevention of autoimmune diseases (33, 41, 67, 70). Although α-GalCer has been reported to enhance host resistance to some microbial pathogens (27-29, 37, 39, 44, 55, 64), its potential role in protection against intracellular bacterial infections remains enigmatic.We have recently described that α-GalCer ameliorates murine listeriosis, which is, in part, caused by accelerated infiltration of inflammatory cells into the liver (18), although iNKT cells themselves exacerbate disease (19). Because Mφ play a central role in the elimination of L. monocytogenes, we considered the possibility that Mφ participate in enhanced resistance to L. monocytogenes infection caused by α-GalCer treatment. In the present study, we examined the influence of α-GalCer on listericidal activities of Mφ using a virulent and an avirulent strain of L. monocytogenes.     

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.     

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.     

Staphylococcus aureus Induces Microglial Inflammation via a Glycogen Synthase Kinase 3β-Regulated Pathway

A proinflammatory role for glycogen synthase kinase 3β (GSK-3β) has been demonstrated. Here, we addressed its roles on heat-inactivated Staphylococcus aureus-induced microglial inflammation. Heat-inactivated S. aureus induced tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, at least in part, via a Toll-like receptor 2-regulated pathway. Neutralization of TNF-α largely blocked heat-inactivated S. aureus-induced NO. Heat-inactivated S. aureus activated GSK-3β, and inhibiting GSK-3β reduced TNF-α production as well as inducible NO synthase (iNOS)/NO biosynthesis. While activation of NF-κB was essential for heat-inactivated S. aureus-induced TNF-α and NO, inhibiting GSK-3β blocked heat-inactivated S. aureus-induced NF-κB p65 nuclear translocation. Additionally, inhibiting GSK-3β enhanced heat-inactivated S. aureus-induced interleukin-10 (IL-10) production (IL-10 is an anti-inflammatory cytokine which inhibits TNF-α production). Neutralization of IL-10 reduced TNF-α downregulation caused by GSK-3β inhibition. These results suggest that GSK-3β regulates heat-inactivated S. aureus-induced TNF-α and NO production in microglia mainly by activating NF-κB and probably by inhibiting IL-10.Staphylococcus aureus, a gram-positive bacterium, causes a variety of diseases, such as bacteremia, peritonitis, subcutaneous and brain abscess, and life-threatening staphylococcal septic shock (15). The mechanisms that lead to staphylococcal septic shock are multifactorial but involve especially immunogenic and toxic injuries (10, 40). Cell wall components and secreted virulence factors, including enterotoxins and exotoxins, have been shown to be inflammatory and cytotoxic to the host. Pathogen-associated molecular patterns are recognized by the innate immune system through a family of pattern recognition receptors, such as Toll-like receptors (TLRs) (2, 6, 26). Microglia, the resident macrophages in the brain, express TLR2 to recognize S. aureus peptidoglycan and play a critical role in neuroinflammation (7, 35, 37). Induction of neuroinflammation by S. aureus is partially mediated by TLR2- and nuclear factor-κB (NF-κB)-regulated pathways (23, 26, 36, 51).Infection of S. aureus causes the deregulated production of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-10, and chemokines, including monocyte chemoattractant protein 1 (MCP-1) and RANTES (regulated on activation, normal T cell expressed and secreted protein) (24, 25, 32, 45). TNF-α, a potent proinflammatory cytokine, causes severe inflammatory responses, including cytokine and chemokine production and inducible nitric oxide (NO) synthase (iNOS)/NO biosynthesis in S. aureus infection (49). The deregulated generation of NO contributes to S. aureus-induced circulatory failure and liver injury (34). IL-10, a potent anti-inflammatory cytokine, inhibits the synthesis of the proinflammatory cytokines (TNF-α, IL-1, IL-6, IL-12, IL-18, and IL-10 itself), chemokines (IL-8, MCP-1, and RANTES), and iNOS/NO (4, 30, 43). IL-10 knockout mice display high mortality and are more susceptible to S. aureus-induced brain abscess (48). Exogenous IL-10 inhibits lethal sepsis, hepatic injury, and TNF-α production induced by staphylococcal enterotoxin B in mice (46, 48).Inhibiting glycogen synthase kinase 3β (GSK-3β) downregulates TLR-mediated inflammatory responses but increases IL-10 production (41, 53). Since NF-κB is important for inflammatory activation, GSK-3β is also involved in activating NF-κB in response to inflammatory stimuli (17-21, 29, 44, 50, 52). Therefore, GSK-3β inhibitors have been used to confer anti-inflammation against TNF-α administration, endotoxemia, experimental colitis, type II collagen-induced arthritis, ovalbumin-induced asthma, and experimental autoimmune encephalomyelitis (5, 12-14, 18, 20, 31, 41, 50, 52). Notably, current studies also show the effects of GSK-3β inhibition in reducing gram-negative coccobacillus Francisella-induced inflammation (55). GSK-3β inhibitors have also been widely used to reduce microglial inflammation and neurotoxicity (31, 54). In search of strategies against S. aureus-induced microglial inflammation, we investigated the possible effects of GSK-3β inhibition. In the present study, we report that inhibiting GSK-3β blocks NF-κB activation, TNF-α production, and iNOS/NO biosynthesis, but increases IL-10 production in heat-inactivated S. aureus-stimulated microglia.     

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-γ) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-γ-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-γ and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-γ and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.The attenuated Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) is the currently used vaccine against tuberculosis (TB). In spite of its wide use, the BCG vaccine only protects against severe forms of childhood TB and generally does not prevent adult pulmonary TB (11, 30, 47).Considering that one-third of the world population is thought to be infected with Mycobacterium tuberculosis and that only a small proportion of these individuals will develop active disease, new vaccine candidates to prevent the establishment of infection could also boost and improve the cellular immunity of already latently infected individuals. Vaccine candidates currently in clinical trials include improved recombinant BCG vaccines, virus-based recombinant vaccines, and subunit vaccines comprised of dominant secreted antigens (1, 32). Secreted proteins, regularly described as culture filtrate proteins (CFP), are the main targets of the T-cell response in mice, both at the height of infection and in a state of memory immunity, as well as in humans with active TB (1, 4, 5, 7, 23). Immunization with these antigens in the presence of different adjuvants provided protection in mice challenged with M. tuberculosis, and protection was mediated by gamma interferon (IFN-γ)-producing CD4⁺ cells (29, 38).We previously showed that a homologous immunization schedule based on three doses of CFP antigens plus CpG oligodeoxynucleotide adjuvant stimulated significant IFN-γ production by spleen cells and in the lungs of challenged mice. In spite of high IFN-γ concentrations, immunized and challenged mice were not protected and indeed had extensive lung damage (16).Since IFN-γ is the best indicator of protective immunity defined thus far, we changed the schedule of homologous immunization to heterologous immunization, also known as a prime-boost regimen, to induce protection.Several studies have demonstrated the efficacy of prime-boost vaccination strategies in generating cellular immunity to a variety of pathogens (3, 10, 14, 17, 34, 36, 44, 45, 49). Recently, our group also showed that a single dose of a DNA-HSP65 vaccine booster significantly enhanced the protection conferred against TB by a single subcutaneous dose of BCG (18). In addition, secreted antigens such as the 6-kDa early-secretion antigen target (ESAT-6), 85A or 85B antigens, and Mtb72F have proven to be promising candidates for BCG-boosting vaccines in mice, guinea pigs, and nonhuman primates (6, 9, 12, 19, 33, 37, 46, 48). Because a single dominant antigen may not confer the same level of protection to all vaccinated individuals, and based on high CFP antigen-mediated IFN-γ production in the presence of CpG adjuvant, in this study we used CFP plus CpG oligodeoxynucleotides to boost BCG vaccination in order to improve protection and lung preservation following M. tuberculosis challenge.     

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.     

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.     

Protection against Intestinal Amebiasis by a Recombinant Vaccine Is Transferable by T Cells and Mediated by Gamma Interferon

We have previously shown that vaccination with purified Entamoeba histolytica Gal/GalNAc lectin or recombinant subunits can protect mice from intestinal amebiasis upon intracecal challenge. In this study, we demonstrated with adoptive-transfer experiments that this lectin vaccine protection is mediated by T cells but not serum. The cell-mediated immune (CMI) response was characterized by significant gamma interferon (IFN-γ), interleukin 12 (IL-12), IL-2, IL-10, and IL-17 production. To move toward a human vaccine, we switched to a recombinant protein and tested a range of adjuvants and routes appropriate for humans. We found that subcutaneous delivery of LecA with IDRI''s adjuvant system EM014 elicited a potent Th1-type CMI profile and provided significant protection, as measured by culture negativity (79% efficacy); intranasal immunization with cholera toxin provided 56% efficacy; and alum induced a Th2-type response that protected 62 to 68% of mice. Several antibody and CMI cytokine responses were examined for correlates of protection, and prechallenge IFN-γ⁺ or IFN-γ-, IL-2-, and tumor necrosis factor alpha-triple-positive CD4 cells in blood were statistically associated with protection. To test the role of IFN-γ in LecA-mediated protection, we neutralized IFN-γ in LecA-immunized mice and found that it abrogated the protection conferred by vaccination. These data demonstrate that CMI is sufficient for vaccine protection from intestinal amebiasis and reveal an important role for IFN-γ, even in the setting of alum.The enteric protozoan parasite Entamoeba histolytica is the causative pathogen of amebic dysentery and liver abscess that affects millions of people worldwide. Bangladeshi children experience a 40% annual incidence of E. histolytica infection (24), and evidence of prior E. histolytica infection can be detected in 8.4% of the general population in Mexico (6). Despite the availability of effective antibiotics, the World Health Organization estimates that up to 100,000 deaths occur annually, highlighting the need for alternate approaches to control amebiasis. One approach is to develop a vaccine to prevent intestinal infection (26).Several vaccine candidates for amebiasis have been proposed (48), including the serine-rich E. histolytica protein, peroxiredoxin, the EhCP112 molecule, and the galactose/N-acetyl-d-galactosamine-inhibitable lectin (Gal/GalNAc lectin). A significant body of work has focused on the latter: vaccination with either parasite-purified Gal/GalNAc lectin (10, 29, 32, 38, 40) or recombinant lectin subunits has provided protection in rodent models against amebic liver abscess and amebic colitis (29, 37, 46, 47, 53). Although these results are encouraging, two limitations remain. First, in most of these vaccine studies, the adjuvants and delivery routes are not compatible with eventual use in humans. Second, the mechanisms of amebiasis vaccine-mediated protection are still not fully understood. For instance, in the intestinal model, there was an association between the presence of an antiparasite lectin fecal immunoglobulin A (IgA) response and subsequent protection, but the association did not extend to the recombinant antigen, and fecal IgA-negative mice remained statistically protected, suggesting that other immune mechanisms exist (29). Indeed, there is increasing evidence for a role of cell-mediated immunity (CMI) in protection from intestinal amebiasis (14, 20, 28). We have found that gamma interferon (IFN-γ), the canonical Th1 cytokine, can clear intestinal amebic infection in CBA mice (21). In this study, we demonstrate for the first time that CMI plays a critical role in lectin-elicited protective immunity to intestinal amebic infection. A variety of vaccine adjuvants and delivery routes were tested for their effectiveness in protection and in eliciting CMI, and the tests demonstrated a clear role for CMI and IFN-γ in lectin-based vaccine protection.     

Cellular Tumor Necrosis Factor,Gamma Interferon,and Interleukin-6 Responses as Correlates of Immunity and Risk of Clinical Plasmodium falciparum Malaria in Children from Papua New Guinea

The role of early to intermediate Plasmodium falciparum-induced cellular responses in the development of clinical immunity to malaria is not well understood, and such responses have been proposed to contribute to both immunity and risk of clinical malaria episodes. To investigate whether P. falciparum-induced cellular responses are able to function as predictive correlates of parasitological and clinical outcomes, we conducted a prospective cohort study of children (5 to 14 years of age) residing in a region of Papua New Guinea where malaria is endemic Live, intact P. falciparum-infected red blood cells were applied to isolated peripheral blood mononuclear cells obtained at baseline. Cellular cytokine production, including production of interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor (TNF) (formerly tumor necrosis factor alpha), and gamma interferon (IFN-γ), was measured, and the cellular source of key cytokines was investigated. Multicytokine models revealed that increasing P. falciparum-induced IL-6 production was associated with an increased incidence of P. falciparum clinical episodes (incidence rate ratio [IRR], 1.75; 95% confidence interval [CI], 1.20 to 2.53), while increasing P. falciparum-induced TNF and IFN-γ production was associated with a reduced incidence of clinical episodes (IRR for TNF, 0.55 [95% CI, 0.38 to 0.80]; IRR for IFN-γ, 0.71 [95% CI, 0.55 to 0.90]). Furthermore, we found that monocytes/macrophages and γδ-T cells are important for the P. falciparum-induced production of IL-6 and TNF. Early to intermediate cellular cytokine responses to P. falciparum may therefore be important correlates of immunity and risk of symptomatic malaria episodes and thus warrant detailed investigation in relation to the development and implementation of effective vaccines.Individuals living in regions of moderate to high malaria endemicity slowly acquire clinical immunity to Plasmodium falciparum throughout their lives in an age- and exposure-dependent manner (3, 29). This immunity enables them to control parasite replication at densities below that which induces clinical symptoms (29, 46). Both antibody-dependent and T-cell-dependent acquired immune responses have been shown to play an important role in the development of clinical immunity (13, 29). The role of early to intermediate cellular responses, however, is less well understood, and such responses have been proposed to contribute to both immunity and risk of clinical malaria episodes (45, 49, 51).Early cellular immune responses are rapidly initiated during malaria infection and are thought to play an important role both in limiting initial parasite replication and in directly shaping subsequent adaptive immune responses (45, 49, 51). However, the overproduction or inappropriate regulation of both proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, gamma interferon (IFN-γ), and tumor necrosis factor (TNF) (formerly tumor necrosis factor alpha), and anti-inflammatory cytokines, such as IL-10, IL-4, and transforming growth factor β (TGF-β), may also lead to localized and systemic inflammation and has been associated with symptomatic and severe malaria (7, 45). The clinical outcome of an infection may thus depend on the appropriate induction and counterregulation of both pro- and anti-inflammatory cytokine secretion. Understanding how this network of P. falciparum-induced cellular responses is associated with immunity and risk of clinical disease in malaria exposed-children could provide important insights for the development of effective vaccines.Studies investigating the association between P. falciparum- or antigen-induced secretion of cytokines from peripheral blood mononuclear cells (PBMCs) and prospective risk of clinical episodes in malaria-exposed individuals provide the most powerful means by which to understand the relationship between these inducible cellular immune responses and risk of disease (8, 19, 29). Two such prospective cohort studies report that late-stage IFN-γ responses to liver-stage and merozoite surface antigens, as well as IL-10 responses to liver-stage antigens, are associated with resistance to reinfection (23, 26). However, stimulation of PBMCs with live, intact P. falciparum-infected red blood cells (iRBCs) at the late trophozoite-schizont stage is thought to more closely reflect the in vivo situation and in malaria-naïve adults has been shown to capture early cellular responses to diverse P. falciparum stimuli (1, 9, 11, 16). For malaria-exposed individuals, Dodoo et al. (8) reported an association between late-stage, live P. falciparum-induced IFN-γ production and reduced risk of fever and clinical malaria. More recently, we reported that early P. falciparum-induced IFN-γ production from γδ-T cells and αβ-T cells is associated with protection from clinical P. falciparum episodes in a cohort of 5- to 14-year-old children from a region of Papua New Guinea (PNG) where malaria is endemic (10).The aims of the present study were to investigate the contribution of multiple P. falciparum-induced early cellular immune responses to immunity and risk of clinical malaria episodes and to evaluate these early cytokine responses together using a multivariate model rather than as isolated responses. A 6-month prospective cohort study was conducted in the Mugil region of PNG (31), and live, intact P. falciparum-iRBCs were applied to isolated PBMCs from 165 malaria-exposed children with ages of 5 to 14 years. Specifically, we sought to determine whether it is possible to define predictive correlates of immunity and risk by examining whether P. falciparum-induced production of cytokines (IL-2, IL-4, IL-6, IL-10, TNF, and IFN-γ) in defined cell populations is associated with age, infection status, and prospective risk of P. falciparum infection and clinical episodes.     

Neutralization of Interleukin-10 from CD14+ Monocytes Enhances Gamma Interferon Production in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Goats

The gamma interferon assay is used to identify Mycobacterium avium subsp. paratuberculosis-infected animals. It has been suggested that regulatory mechanisms could influence the sensitivity of the test when it is performed with cells from cattle and that the neutralization of interleukin-10 (IL-10) in vitro would increase the gamma interferon responses. To investigate the regulatory mechanisms affecting the gamma interferon assay with cells from goats, blood was collected from M. avium subsp. paratuberculosis-infected, M. avium subsp. paratuberculosis-exposed, and noninfected goats. Neutralization of IL-10 by a monoclonal antibody resulted in increased levels of gamma interferon production in M. avium subsp. paratuberculosis purified protein derivative (PPDj)-stimulated samples from both infected and exposed goats. However, the levels of gamma interferon release were also increased in unstimulated cells and in PPDj-stimulated cells from some noninfected animals following neutralization. Depletion of putative regulatory CD25^high T cells had no clear effect on the number of gamma-interferon-producing cells. The IL-10-producing cells were identified to be mainly CD14⁺ major histocompatibility complex class II-positive monocytes in both PPDj-stimulated and control cultures and not regulatory T cells. However, possible regulatory CD4⁺ CD25⁺ T cells produced IL-10 in response to concanavalin A stimulation. The numbers of CD4⁺, CD8⁺, and CD8⁺ γδT-cell receptor-positive cells producing gamma interferon increased following IL-10 neutralization. These results provide insight into the source and the role of IL-10 in gamma interferon assays with cells from goats and suggest that IL-10 from monocytes can regulate both innate and adaptive gamma interferon production from several cell types. Although IL-10 neutralization increased the sensitivity of the gamma interferon assay, the specificity of the test could be compromised.Mycobacterium avium subsp. paratuberculosis is the causative agent of paratuberculosis, a chronic intestinal disease in ruminants (12) that has a worldwide distribution and that is of substantial economic importance (5, 29). To control paratuberculosis, it is of great importance to be able to identity infected animals at an early stage. Several methods can be used to diagnose paratuberculosis, but all of these methods have limitations. Fecal culture, PCR, and serum antibody assays have relatively low sensitivities in the early stages of the disease (2, 44), while the gamma interferon (IFN-γ) test has the greatest potential to detect paratuberculosis in subclinically infected animals (7, 37). Norwegian goat kids experimentally infected with high doses of M. avium subsp. paratuberculosis gave increased IFN-γ responses from week 7 after the first bacterial exposure (40), and the results of an evaluation of the test for the diagnosis of paratuberculosis in Norwegian goats was promising (39). However, data from a Norwegian surveillance program have suggested that positive IFN-γ results are not detected earlier than antibodies in blood or bacteria in feces in naturally infected goats (K. R. Lybeck, unpublished data).IFN-γ production is induced by cytokines like interleukin-12 (IL-12) and IL-18, while IL-4 and IL-10 reduce the level of IFN-γ expression (13, 30, 34). IL-10 can limit IFN-γ production in M. avium subsp. paratuberculosis- or Mycobacterium bovis-infected cattle, and it has been suggested that the neutralization of IL-10 could be a way of increasing the sensitivity of the IFN-γ assay since this leads to an antigen-specific increase in the level of IFN-γ production (10, 15). The cells producing IL-10 in those studies were not identified, but it was speculated that regulatory T cells (Tregs) were involved (15). Lately, the role of Tregs in suppressing immune responses has been a focus, and regulatory IL-10-producing CD4⁺ CD25⁺ cells from M. avium subsp. paratuberculosis-infected cattle have been reported (14).In humans and mice, Tregs control the immune responses to infections, balancing protective immunity and immunopathology. However, during mycobacterial infections as well as infections caused by some other organisms, it seems that the suppression of the protective immune response can lead to pathogen persistence and chronic disease (21, 25, 26). Both thymus-derived natural CD4⁺ CD25^high Foxp3 Tregs and adaptive CD4⁺ CD25^+/− Foxp3^+/− Tregs induced outside the central lymphoid organs exist (8, 35). It is generally believed that natural Tregs mediate suppression through contact-dependent mechanisms, while adaptive Tregs act via production of the anti-inflammatory cytokine IL-10 or transforming growth factor β (TGF-β) (8, 26). IL-10 is also produced by macrophages, dendritic cells, B cells, and various subsets of CD4⁺ and CD8⁺ T cells; and excessive or mistimed IL-10 production can inhibit protective immune responses to intracellular pathogens (13). TGF-β is produced by most immune cells and has the potential to suppress IFN-γ production (11).On the basis of the knowledge of regulatory T cells in humans and mice and the findings from studies with cattle linking suboptimal IFN-γ production to regulatory mechanisms, we hypothesized that the low sensitivity of the IFN-γ test observed with cells from subclinically paratuberculosis-infected goats could be due to the effect of regulatory mechanisms. These mechanisms are poorly described in goats, and the aim of this study was to investigate the regulatory factors influencing the IFN-γ responses to paratuberculosis in vitro and ultimately identify possible ways to increase the sensitivity of the IFN-γ assay.     

Mitogenic component in polar lipid-enriched Anaplasma phagocytophilum membranes

Human granulocytic anaplasmosis is an emerging tick-borne disease caused by Anaplasma phagocytophilum. A. phagocytophilum cells activate Toll-like receptor 2 signaling and possess mitogenic activity, and A. phagocytophilum infection in vivo activates NKT cells unrelated to major surface protein 2 (Msp2) hypervariable region expression. Thus, we hypothesized that lipoprotein or glycolipid components of A. phagocytophilum membranes could be important triggers of the innate immune response and immunopathology. A. phagocytophilum membranes depleted of Msp2 and protein antigens enhanced the proliferation of naïve mouse splenocytes beyond that of untreated membranes. Protein-depleted and polar lipid-enriched membranes from low-passage A. phagocytophilum cultures enhanced naïve splenocyte lymphoproliferation to a much greater degree than did these fractions from high-passage cultures of bacterial membranes (1.8- to 3.7-fold for protein-depleted fractions and 4.8- to ≥17.7-fold for polar lipid-enriched fractions). These results support the hypothesis that components that are enriched among polar lipids in the A. phagocytophilum membrane stimulate innate immune cell proliferation, possibly activating NKT cells that link innate and adaptive immunity, and immunopathology.Human granulocytic anaplasmosis (HGA) is an emerging tick-borne disease caused by Anaplasma phagocytophilum, an obligate intracellular bacterium that infects neutrophils and lacks pathways for both lipopolysaccharide (LPS) and peptidoglycan biosynthesis (10, 16). Although the majority of human infections are mild, severe and even fatal infections complicated by interstitial pneumonitis, adult respiratory distress syndrome, and sepsis or toxic shock-like syndromes are well recognized (10, 20, 22, 30). The inflammatory basis of the disease is demonstrated in human histopathologic investigations and animals, including mouse models (4, 13, 15). In the mouse model, pathogenicity results from the induction of innate immunity and gamma interferon (IFN-γ), which drives local effectors and inflammatory tissue injury (3, 17, 18, 26). However, this inflammatory phenotype is also determined in part by bacterial factors, since infection of horses by isogenic A. phagocytophilum differing only in the length of in vitro passage results in differential clinical severity and hematologic derangements (21). A similar dichotomous phenotype was observed among B6 mice infected with A. phagocytophilum propagated for different lengths of time in vitro, resulting in the differential expansion of NK1.1⁺ cells and CD8⁺ splenic lymphocytes on days 2 to 7 (9). In fact, peak hepatic inflammation and plasma IFN-γ production with infection by low-passage bacteria occur on day 2, when adaptive immunity is very unlikely to be present, corresponding to approximately the same time that splenic NKT cells become activated.T-cell responses to the immunodominant major surface protein 2 (Msp2) hypervariable regions that vary with in vitro propagation do not occur to any substantial degree, diminishing their importance as inflammatory stimuli (7). However, lymphoproliferative responses to whole-cell A. phagocytophilum cultures are detectable even among splenocytes from naïve mice. Owing to these data, the innate immune responses in mice infected with A. phagocytophilum, and our prior data implicating Toll-like receptor 2 (TLR2) but not TLR4 inflammatory signaling in human and murine macrophages exposed to A. phagocytophilum (8, 9, 26), we hypothesized that a lipoprotein or glycolipid component of A. phagocytophilum membranes is an important trigger of the innate immune response and immunopathology. Thus, we studied A. phagocytophilum to identify whether bacterial membranes and membrane components could initiate differential naïve immune cell proliferation that in part underlies the virulence observed with changing in vitro passage intervals.     

Yersinia enterocolitica Promotes Robust Mucosal Inflammatory T-Cell Immunity in Murine Neonates

Mucosal immunity to gastrointestinal pathogens in early life has been studied only slightly. Recently, we developed an infection model in murine neonates using the gastroenteric pathogen Yersinia enterocolitica. Here, we report that oral infection of neonatal mice with low doses of virulent Y. enterocolitica leads to vigorous intestinal and systemic adaptive immunity. Y. enterocolitica infection promoted the development of anti-LcrV memory serum IgG1 and IgG2a responses of comparable affinity and magnitude to adult responses. Strikingly, neonatal mesenteric lymph node CD4⁺ T cells produced Yersinia-specific gamma interferon (IFN-γ) and interleukin-17A (IL-17A), exceeding adult levels. The robust T- and B-cell responses elicited in neonates exposed to Y. enterocolitica were associated with long-term protection against mucosal challenge with this pathogen. Using genetically deficient mice, we found that IFN-γ and CD4⁺ cells, but not B cells, are critical for protection of neonates during primary Y. enterocolitica infection. In contrast, adults infected with low bacterial doses did not require either cell population for protection. CD4-deficient neonatal mice adoptively transferred with CD4⁺ cells from wild-type, IFN-γ-deficient, or IL-17AF-deficient mice were equally protected from infection. These data demonstrate that inflammatory CD4⁺ T cells are required for protection of neonatal mice and that this protection may not require CD4-derived IFN-γ, IL-17A, or IL-17F. Overall, these studies support the idea that Y. enterocolitica promotes the development of highly inflammatory mucosal responses in neonates and that intestinal T-cell function may be a key immune component in protection from gastrointestinal pathogens in early life.Host protection against microbial agents ultimately relies on the cooperative action of the innate and adaptive immune systems. In both human and murine neonates, adaptive immune responses are compromised compared to responses in developmentally mature hosts (5, 66). Factors that may contribute to the immunological immaturity reported during neonatal life include the following: the lack of antigen-specific immunological memory (5, 65), reduced levels of antigen presenting cells (APC) (46) and adaptive immune cells (21), delays in the development of lymph node germinal centers (57), and cell-intrinsic differences in immune responsiveness (4, 48, 67). Thus, neonatal immune responses following infection or vaccination often appear to be diminished compared to responses in adults. In particular, B-cell and CD4⁺ T helper (T_h) responses to a variety of antigens may be reduced in magnitude, quality, and duration (5, 65). Neonatal immunization with prototypic protein vaccine antigens often leads to mixed T_h1 and T_h2 primary responses (2), but the development of T_h1-associated memory (3) and production of T_h1-associated IgG2a antibodies are often reduced compared to these responses in adults (9). However, adult-like T_h1 immunity has been achieved in neonatal hosts after Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination (53, 72), DNA vaccines (55, 62), or attenuated vaccinia-derived vectors (44). These observations led to the recognition that immune responsiveness during early life could be greatly enhanced by optimizing the conditions of antigen exposure using highly inflammatory treatments.Activation of the neonatal immune system through microbe-associated molecular pattern receptors has demonstrated remarkable improvements in promoting effective immunity to vaccine antigens. For example, bacterially derived products such as mutated Escherichia coli enterotoxins LT-R192G (70) and LT-K63 (11, 14, 27, 34), CpG oligonucleotides (CpG) (8, 29, 31), and lyophilized bacterial extracts (12) have been described to markedly enhance neonatal vaccine responses. Another approach used to improve immune responses has been the delivery of specific antigens using live attenuated bacterial vectors such as Listeria monocytogenes (42, 50) and Salmonella species (16, 59). Both of these approaches have shown dramatic improvements in CD4⁺ and CD8⁺ IFN-γ production, mucosal IgA production, and systemic IgG1 and IgG2a antibodies to the delivered vaccine antigens. Recently, CD4⁺ T_h17-mediated immunity has been studied in response to vaccination with rotavirus antigen in adjuvant (70) and to Mycobacterium tuberculosis antigens in the presence of non-CpG oligonucleotides (36) or cationic liposomes (37). These vaccines promoted interleukin-17A (IL-17A) levels of the same magnitude in neonatal and adult CD4⁺ cells (36, 37, 70). Altogether, it has become apparent that under the proper stimulation conditions, all arms of the neonatal adaptive immune system can be induced to generate adult-like responses. Importantly, some of these immunization regimens promoted protective immunity against infection with fully pathogenic bacteria (16, 29, 31, 34, 42, 59).Despite the profound maturation of the neonatal immune system through vaccination with live attenuated L. monocytogenes and Salmonella vectors (16, 17, 42, 50, 59), neonatal immune responses to fully virulent pathogens are inefficient in controlling infection (15, 25, 31, 61). This exquisite susceptibility to infection during neonatal life includes both peripheral and mucosal routes of infection. In particular, neonatal animals succumb rapidly to pulmonary infection with Streptococcus pneumoniae (24) and gastrointestinal infection with enteropathogens including Vibrio cholerae (10), Aeromonas hydrophila (76), Shigella flexneri (23), enterotoxigenic E. coli (19), and Salmonella species (15, 61). Thus, mucosal immune responses to most pathogens studied to date are severely compromised in early life.In contrast to the vast majority of experimental systems, we recently demonstrated (20) that murine neonates are highly resistant to oral infection with the Gram-negative enteropathogen Yersinia enterocolitica. The resistance of neonatal mice infected with Y. enterocolitica was associated with robust innate inflammation, characterized by the recruitment of high levels of neutrophils and macrophages into the intestinal tissue (20). We hypothesized that the vigorous innate responses in neonates may promote similarly robust adaptive immunity. Here, we have compared the development of Yersinia-specific B- and CD4⁺ T-cell immunity in neonatal and adult mice. We demonstrate that highly protective intestinal and systemic adaptive immunity can be induced in neonatal mice. Remarkably, neonatal mice developed greater Yersinia-specific T_h1 and T_h17 responses in the mesenteric lymph nodes (MLN) than did adults. Experiments using genetically deficient mice with or without adoptive transfer of donor cells showed that CD4⁺ T cells, but not B cells, appeared to be necessary for resistance of infected neonates. Thus, we extend our earlier studies to further demonstrate the unprecedented inflammatory potential of the neonatal gastrointestinal immune system in response to a fully virulent enteric pathogen.     

Multiple T-Cell Responses to Human Immunodeficiency Virus Type 1 Are Enhanced by Dendritic Cells

Human immunodeficiency virus type 1 (HIV-1)-specific T-cell reactivity has been related to protection from disease progression. Optimal T-cell reactivity to HIV-1 presumably requires antigen processing and presentation by professional antigen-presenting cells, particularly dendritic cells (DC). Here we examined whether multiple HIV-1-specific T-cell functions are enhanced by stimulation with HIV-1 peptide-loaded DC derived from HIV-1-infected subjects on antiretroviral therapy. We first found that mature DC increased the number of gamma interferon (IFN-γ)-producing T cells detected by enzyme-linked immunospot assay to overlapping 15-mer peptides of HIV-1 Gag and Nef, compared to stimulation with peptide-loaded, immature DC or to peptides without DC. IFN-γ production was lower in response to large pools of the Gag and Nef peptides, regardless of presentation by DC. We further observed that HIV-1 peptide-loaded, mature DC stimulated greater CD8⁺ and CD4⁺ T-cell proliferation than did the peptides without DC and that T-cell proliferation was lower in response to larger pools of the peptides. The lower T-cell IFN-γ and proliferation responses to the larger peptide pools were related to lower T-cell viability. Finally, the number of polyfunctional CD8⁺ and CD4⁺ T cells stimulated by HIV-1 peptide-loaded, mature DC, defined as positive by intracellular staining for more than one immune mediator (IFN-γ, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1β, or CD107a), was greater than that stimulated by the peptides alone. These results indicate that DC can enhance multiple types of HIV-1-specific T-cell functions.Considerable evidence supports the idea that T-cell immunity to human immunodeficiency virus type 1 (HIV-1) is important in control of HIV-1 infection (10). Specific correlates of T-cell immunity that are associated with protection against or progression of HIV-1 infection have nonetheless been difficult to determine. Such immune correlates could be useful in defining the efficacy of prophylactic and therapeutic vaccines for HIV-1 infection. Many studies of T-cell immunity in HIV-1 infection have shown that the number of T cells exhibiting gamma interferon (IFN-γ) production in the enzyme-linked immunospot (ELISPOT) assay is decreased in association with progressive infection (4, 51). Proliferation of T cells in response to HIV-1 antigens as measured by uptake of the succinimidyl ester of carboxyfluorescein diacetate (CFSE) has also been related to less progressive HIV-1 infection (19, 33, 53). Recently, the quality of the CD8⁺ T-cell functional response to HIV-1 peptides as defined by intracellular cytokine staining (ICS) for more than one immune mediator, i.e., IFN-γ, interleukin 2 (IL-2), tumor necrosis factor alpha (TNF-α), macrophage inhibitory protein 1β (MIP-1β), and/or cytotoxic degranulation molecule CD107a (11, 44), has been associated with slow progression and better control of HIV-1 infection (5).Although these are all valid measures of anti-HIV-1 T-cell immunity, they usually do not account for a role of professional antigen-presenting cells (APC), particularly dendritic cells (DC), which are necessary for optimal processing and presentation of antigens to T cells (2). Indeed, it is likely that during HIV-1 infection, DC are required to take up, process, and present HIV-1 antigens via their major histocompatibility complex (MHC) class I and II molecules for priming and boosting of anti-HIV-1 CD8⁺ and CD4⁺ T-cell responses (40). We have previously shown that IFN-γ production by CD8⁺ T cells from HIV-1-infected persons is enhanced by stimulation with DC loaded with HIV-1 antigens and matured with CD40L or a cocktail of various proinflammatory cytokines and a Toll-like receptor 3 ligand (15, 20, 21). Myeloid DC loaded with peptides representing dominant epitopes of HIV-1 proteins stimulated significantly more epitope-specific, IFN-γ-producing CD8⁺ T cells than did peptides added directly to peripheral blood mononuclear cells (PBMC). There is little information, however, as to whether these professional APC can similarly enhance other T-cell functions that could be critical to control of HIV-1 infection, particularly their proliferative capacity and ability to produce multiple immune mediators. Moreover, many current approaches for measuring the magnitude and breadth of T-cell responses use pools of various numbers of synthetic peptides, usually 15 or 20 amino acids (aa) in length, which overlap by 10 to 11 aa (1, 3, 7, 9, 13, 14, 17, 24, 25, 27, 32, 37, 45, 48, 49), developed by Kern et al. (26) and Maecker et al. (31). Such studies have not accounted for a role of APC in processing that is required to reduce these peptides to their optimal, 8- to 10-mer length for presentation by MHC class I molecules to CD8⁺ T cells (43), or to 13- to 17-mers for presentation by MHC class II to CD4⁺ T cells (46). These are important considerations in determining correlates of T-cell immunity in HIV-1 infection and in response to HIV-1 vaccines.We have analyzed the magnitude of several types of T-cell responses during HIV-1 infection stimulated by autologous DC loaded with different-size pools of overlapping HIV-1 peptides. We assessed T-cell responses in HIV-1- infected persons for single-cell IFN-γ production by using a conventional ELISPOT assay; for CD8⁺ and CD4⁺ T-cell proliferation by using uptake of CFSE dye; and for production of IFN-γ, IL-2, TNF-α, MIP-1β, and CD107a by CD8⁺ and CD4⁺ T cells by using ICS. We found that, in addition to enhancing IFN-γ production detected by ELISPOT assay, DC loaded with HIV-1 peptide singlets or smaller pools of HIV-1 peptides enhanced HIV-1-specific T-cell proliferation and polyfunctional CD8⁺ and CD4⁺ T-cell responses.