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.     

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.     

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.     

Helicobacter pylori Stimulates Dendritic Cells To Induce Interleukin-17 Expression from CD4+ T Lymphocytes

Helicobacter pylori is a human gastroduodenal pathogen that leads to active chronic inflammation characterized by T-cell responses biased toward a Th1 phenotype. It has been accepted that H. pylori infection induces a Th17 response. At mucosal sites, dendritic cells (DCs) have the capacity to induce effector T cells. Here, we evaluate the role of DCs in the H. pylori-induced interleukin-17 (IL-17) response. Immunohistochemistry and immunofluorescence were performed on human gastric mucosal biopsy samples and showed that myeloid DCs in H. pylori-infected patients colocalized with IL-23- and that IL-17-producing lymphocytes were present in H. pylori-infected antral biopsy samples. In parallel, human monocyte-derived DCs stimulated in vitro with live H. pylori cells produced significant levels of IL-23 in the absence of IL-12 release. The subsequent incubation of H. pylori-infected DCs with autologous CD4⁺ T cells led to gamma interferon (IFN-γ) and IL-17 expression. The inhibition of IL-1 and, to a lesser extent, IL-23 inhibited IL-17 production by T cells. Finally, isogenic H. pylori mutant strains not expressing major virulence factors were less effective in inducing IL-1 and IL-23 release by DCs and IL-17 release by T cells than parental strains. Altogether, we can conclude that DCs are potent inducers of IL-23/IL-17 expression following H. pylori stimulation. IL-1/IL-23 as well as H. pylori virulence factors seem to play an important role in mediating this response.Gram-negative Helicobacter pylori is a gastroduodenal pathogen identified as being the causative agent of a variety of disease including gastritis, peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoma (23, 27, 41, 42). H. pylori infection of gastric mucosa leads to active chronic inflammation characterized by both a lymphocytic and neutrophil infiltrate with the induction of proinflammatory cytokines, mainly interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), IL-8, and IL-6 (13, 29).The H. pylori-specific gastric mucosal T-cell response is predominantly a CD4⁺ T-cell response polarized toward a T-helper 1 (Th1) phenotype with increased levels of gamma interferon (IFN-γ) (4, 38, 55). Although profound, this immune response does not clear the bacteria, and indeed, the cytokines secreted are more associated with pathogenesis (38, 45). Furthermore, neutrophil responses are associated with tissue damage and ulceration (7, 60). The release of the neutrophil chemoattractant IL-8 by gastric epithelial cells was previously shown to depend on the expression of an H. pylori virulence factor: the cytotoxin-associated gene (cag) pathogenicity island (PAI) (14, 62). The cag PAI encodes the immunodominant protein CagA and the type IV secretion system, which serves to transfer the bacterial CagA protein and other soluble factors, such as peptidoglycans, to the cytoplasm of the host cell (9, 52). Strains expressing the cag PAI have been associated with a more severe inflammatory response than that induced by cag PAI-negative strains (12). The cellular recognition of cag PAI-positive strains is mediated via signaling through the host-intracellular pathogen recognition molecule NOD1 (nucleotide-binding oligomerization domain 1), leading to NF-κB activation and the induction of proinflammatory responses (58).It was previously shown that H. pylori infection is also associated with a marked production of Th17 cytokines (2, 39, 44). By using real-time PCR and Western blotting, it was previously demonstrated that IL-17, a proinflammatory cytokine, is upregulated in H. pylori-infected stomach biopsy specimens in comparison to uninfected specimens (39). IL-17 is a cytokine that characterizes a distinct population of T cells, namely, Th17 (1, 28). IL-17 has been associated with chronic inflammatory conditions such as rheumatoid arthritis (10) and multiple sclerosis (37). In addition, IL-17 proinflammatory function leading to IL-8 stimulation raises the possibility that IL-17 may play a role during bacterial infections (39, 57). Major cytokines associated with the differentiation of human Th17 cells were identified to be IL-23, IL-1β, and IL-6 (11, 61). While IL-12 plays a key role in the differentiation of naïve T cells to Th1 cells, IL-23 promotes the expansion of Th17 cells. In contrast, IL-27, another IL-12 family member, has been shown to limit the development of Th17 cells (25). IL-12 and IL-23 are heterodimers with a shared subunit, p40. Both IL-23 and IL-12 are produced by activated antigen-presenting cells (APCs) such as DCs and macrophages (48, 53).DCs, which play a central role in the induction of adaptive immune responses, are widely distributed in tissues, including gastrointestinal mucosa (32, 33), and were previously shown to be capable of migrating through epithelial tight junctions to gain access to the gastrointestinal lumen (33, 49). Furthermore, we and others have shown that H. pylori interactions with DCs trigger maturation and activation events that lead to the production of cytokines, which are important for the induction and regulation of immune responses (5, 18, 34, 43, 46).Previous studies of DC activation by H. pylori have focused on the induction of the Th1-biased response. Much less is known about the mechanism of induction as well as the cells and cytokine stimuli responsible for the expression of IL-17 in Helicobacter infection. Here, we have reevaluated the role of DCs in the induction of immune responses to Helicobacter infection by addressing the interaction of H. pylori-infected DCs with CD4⁺ T lymphocytes in initiating a Th17 response.     

Avirulent Uracil Auxotrophs Based on Disruption of Orotidine-5′-Monophosphate Decarboxylase Elicit Protective Immunity to Toxoplasma gondii

The orotidine-5′-monophosphate decarboxylase (OMPDC) gene, encoding the final enzyme of the de novo pyrimidine biosynthesis pathway, was deleted using Toxoplasma gondii KU80 knockouts to develop an avirulent nonreverting pyrimidine auxotroph strain. Additionally, to functionally address the role of the pyrimidine salvage pathway, the uridine phosphorylase (UP) salvage activity was knocked out and a double knockout of UP and OMPDC was also constructed. The nonreverting ΔOMPDC, ΔUP, and ΔOMPDC ΔUP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The ΔUP knockout strain was replication competent and virulent. In contrast, the ΔOMPDC and ΔOMPDC ΔUP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the ΔOMPDC strain but not the ΔOMPDC ΔUP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the ΔOMPDC and ΔOMPDC ΔUP knockout strains exhibited extreme attenuation in murine virulence (∼8 logs). Genetic complementation of the ΔOMPDC strain using a functional OMPDC allele restored normal replication and type I parental strain virulence phenotypes. A single immunization of mice with either the live critically attenuated ΔOMPDC strain or the ΔOMPDC ΔUP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting ΔOMPDC and ΔOMPDC ΔUP strains provide new tools for the dissection of the host response to infection and are promising candidates for safe and effective Th1 vaccine platforms that can be easily genetically engineered.Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates in a wide variety of cell types. Infections are widespread in humans, and while infections in healthy individuals are typically asymptomatic, severe disease can occur in utero or in individuals with severe immune suppression (22, 29, 31). A chronic infection is established and is characterized by quiescent cysts containing bradyzoites in tissues such as brain, muscle, and eye (42). Chronic infection can reactivate in AIDS and cause toxoplasmic encephalitis (9, 31) or recurrent ocular toxoplasmosis, recently recognized as a prevalent retinal infection in the United States (23, 27). Current treatments are poorly tolerated and are ineffective against chronic stages of infection, and there are no vaccines. Targeting of the T. gondii de novo pyrimidine synthesis pathway is one potential approach to developing more-effective vaccination strategies based on live attenuated strains with defined genetic disruptions (14).The key uracil phosphoribosyltransferase (UPRT) activity in the pyrimidine salvage pathway can easily be disrupted, and loss of UPRT has no apparent effect on parasite growth in vitro or virulence in vivo (4, 8). In the absence of any pyrimidine salvage pathway, T. gondii still possesses a complete six-step pathway for the de novo biosynthesis of UMP, the precursor molecule of all essential pyrimidines (1, 14, 34, 38). Insertional disruption of the first step of the biosynthetic pathway, encoded by the carbamoyl phosphate synthetase II (CPSII) gene, produced a severe uracil auxotrophy exemplified by the cps1-1 strain of T. gondii, which was incapable of de novo pyrimidine synthesis (13, 16). After invasion of a host cell, the cps1-1 uracil auxotrophic mutant was starved for pyrimidines and ceased to proliferate, since uracil is not readily available for salvage in mammals (12, 13, 32). The cps1-1 mutant strain also exhibited an extreme attenuation of virulence in both immune-competent and severely immune-deficient homozygous gamma interferon (IFN-γ) knockout mice (13).Immunization of mice with the live attenuated type I cps1-1 strain elicits a potent CD8⁺ T-cell-dependent lifelong protective immunity against infection with type I strains (13, 18) and against infection with type II strains and chronic infection (19). In contrast, immunization with T. gondii extracts or killed noninvasive intact parasites does not elicit significant immunity to reinfection with T. gondii (2, 40). Only actively infected host cells have been shown to prime CD8⁺ T-cell-dependent immunity in T. gondii infection (10, 20, 21).Immunity is effectively elicited by immunization with the cps1-1 strain in C57BL/6 (18), BALB/c (13), and tyk2^−/− signaling-deficient mice (39) and surprisingly also in MyD88^−/− deficient mice (41). Remarkably, macrophages primed in vivo by cps1-1 immunization but not naive macrophages also exhibit extremely efficient ex vivo IFN-γ-mediated innate cellular immunity augmenting intracellular rupture and clearance of type II and type III strains of T. gondii (30, 45-47), whereas virulent type I strains resist this cps1-1-induced innate killing mechanism (46).The lifelong immunity elicited by vaccination with strain cps1-1 is dependent on CD8⁺ T cells and interleukin 12 (IL-12) (41, 44) and is also dependent on IFN-γ (13, 18), although systemic IFN-γ is not required for priming (18). Surprisingly, cps1-1 rapidly elicits functional IL-12p70 both locally and systemically following vaccination (18). In contrast to current models of viral or intracellular bacterial infections, CD8⁺ T-cell-intrinsic IL-12 signaling is required for development of IFN-γ-producing CD8⁺ cytotoxic-T-lymphocyte populations and for the generation of memory CD8⁺ T cells in response to cps1-1 (43, 44). Vaccination with cps1-1 induced four distinct effector CD8⁺ T-cell types based on KLRG1 and CD62L expression levels (44). The rapidly elicited and abundant populations of antigen-specific effector CD8⁺ T cells induced by vaccination with cps1-1 are cytolytic in vitro and in vivo (28). The nonreplicating cps1-1 vaccine model has significantly advanced the understanding of the host response, innate immunity, and protective adaptive immunity to T. gondii infection (3, 10, 13, 18, 19, 28, 30, 39, 41, 43-47).The cps1-1 strain is not easily amenable to further genetic manipulation, and this strain exhibits an extremely low frequency of reversion to the virulent parental (strain RH) phenotype (13). To further address the potential of the six-step pyrimidine synthesis pathway to develop improved avirulent, nonreverting, genetically defined, and genetically manipulatable strains, we deleted the sixth and final enzyme of the pathway by targeted disruption of the orotidine-5′-monophosphate (OMP) decarboxylase, the OMPDC gene, in the RH strain KU80 knockout background, which now enables highly efficient gene targeting (17, 24). Targeted disruption of OMPDC via double-crossover homologous recombination induced severe pyrimidine auxotrophy and resulted in the generation of nonreverting strains that were essentially completely attenuated in their virulence in mice. A single immunization with ΔOMPDC knockout strains effectively induced a potent protective immunity to subsequent lethal challenge infection with T. gondii.     

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.     

Critical Role for Interleukin-1β (IL-1β) during Chlamydia muridarum Genital Infection and Bacterial Replication-Independent Secretion of IL-1β in Mouse Macrophages

Recent findings have implicated interleukin-1β (IL-1β) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1β is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1β expression during a chlamydial genital infection. In the present study, IL-1β^−/− mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1β both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1β secretion in WT mice, the major producers of IL-1β in vivo are F4/80⁺ macrophages and GR-1⁺ neutrophils, but not CD45⁻ epithelial cells. Although elicited mouse macrophages infected with Chlamydia muridarum in vitro secrete minimal IL-1β, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichia coli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1β from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1β secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1β secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1β secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1β mRNA observed in vivo in this cell type.The obligate intracellular pathogen Chlamydia trachomatis is the most common cause of sexually transmitted infection worldwide. Infection can lead to oviduct pathology and infertility in females. Cells respond to infection with C. trachomatis by upregulating a wide assortment of genes, including those for proinflammatory cytokines such as tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6 (34, 54, 55, 68). The cellular paradigm of chlamydial pathogenesis states that tissue damage resulting from chlamydial infection is caused by excessive production of these proinflammatory cytokines (61). In support of this theory, caspase-1 knockout (KO) and Toll-like receptor 2 (TLR2) KO mice exhibit less upper genital tract pathology than wild-type (WT) mice, despite equivalent courses of infection (12, 15). This finding is noteworthy because caspase-1 KO mice lack the protease required to activate IL-1β (32) and TLR2 KO fibroblasts express less mRNA for IL-1β and lower levels of other inflammatory cytokines than WT fibroblasts during in vitro chlamydial infections (15). Additionally, administration of an IL-1β antagonist prevents C. trachomatis-induced cytotoxicity in a human fallopian tube organ culture model (30). Overall, these observations suggest that IL-1β may be a potentially important factor in the development of oviduct pathology, underscoring the need to define the role of IL-1β during in vivo chlamydial infections and to mechanistically determine how the IL-1β-converting enzyme caspase-1 is activated upon contact with Chlamydia spp. to produce mature IL-1β.Caspase-1 is the central component of the host “inflammasome” (reviewed in reference 38) and exists at rest as a 45-kDa zymogen. When activated by proteolysis, it forms a tetramer consisting of two 10- to 20-kDa heterodimers (65), with the 20-kDa portion containing the active-site cysteine necessary to cleave the cytokine precursors pro-IL-1β and pro-IL-18 and also the TH2 cytokine precursor pro-IL-33 (10, 24, 59, 63). Caspase-1 has many other substrates in the cell (60), but its two main targets relevant to inflammation are IL-1β (10) and IL-18 (24). Both IL-1β and IL-18 require this processing for biologic activity. IL-18 plays a role in stimulating gamma interferon production from T cells (18, 56) and natural killer cells (11), suggesting a protective role for IL-18 during genital chlamydial infection. This leads to the theory that the detrimental role of caspase-1 for oviduct pathology during chlamydial infection is mediated mainly via overproduction of the proinflammatory cytokine IL-1β.Several studies have shown that caspase-1 activation during intracellular bacterial infection involves active contribution from the bacteria. For instance, activation occurs following bacterial type III secretion (T3S)-dependent introduction into the cytosol of Yersinia pestis YopJ (35), Pseudomonas aeruginosa flagellin (42), or Salmonella enterica flagellin (20). Early work examining the role of caspase-1 during chlamydial infection by using HeLa cells demonstrated that the activation of caspase-1 late in the infection cycle (36 to 48 h postinfection) is dependent on both chlamydial viability and protein synthesis (36). Additionally, the T3S antagonist INP0007 prevents activation of caspase-1 in C. trachomatis-infected HeLa cells (66). Low-level production of IL-1β was abolished in caspase-1 KO mouse peritoneal macrophages infected in vitro with mouse chlamydia C. muridarum (12), which can replicate effectively in mouse macrophages (46). However, it is not known if the infected epithelial cells or the inflammatory cells recruited to the site of infection are the main producers of IL-1β during an in vivo genital infection. Furthermore, the specific role for IL-1β during in vivo genital infection has not been addressed. In this study, we show that IL-1β is required for optimal chlamydial clearance but also contributes to the development of genital tract pathology. Additionally, macrophages and neutrophils and not epithelial cells account for the high expression levels of IL-1β in vivo. We further describe mechanistic details of IL-1β production by using in vitro infections of lipopolysaccharide (LPS)-primed macrophages. Overall, these macrophages produced IL-1β by both caspase-1- and serine protease-dependent mechanisms, but surprisingly, IL-β secretion by these cells occurred in a bacterial protein synthesis-independent manner, although chlamydial viability was required.     

Critical Role of the Interleukin-17/Interleukin-17 Receptor Axis in Regulating Host Susceptibility to Respiratory Infection with Chlamydia Species

The specific contribution of interleukin-17/interleukin-17 receptor (IL-17/IL-17R)-mediated responses in regulating host susceptibility against obligatory intracellular Chlamydia infection was investigated in C57BL/6 and C3H/HeN mice during Chlamydia muridarum respiratory infection. We demonstrated that Chlamydia stimulated IL-17/IL-17R-associated responses in both Chlamydia-resistant C57BL/6 and Chlamydia-susceptible C3H/HeN mice. However, C3H/HeN mice developed a significantly greater IL-17/IL-17R-associated response than C57BL/6 mice did. This was reflected by an increase in IL-17 mRNA expression, a higher recall IL-17 production from splenocytes upon antigen restimulation, and higher production of Th17-related cytokines (IL-23 and IL-6) and chemokines (chemokine [C-X-C motif] ligand 2 [CXCL1]/keratinocyte-derived chemokine [KC] and CXCL2/macrophage inflammatory protein 1 [MIP2]) in C3H/HeN mice than in C57BL/6 mice. Furthermore, C3H/HeN mice displayed a massive accumulation of activated and preactivated neutrophils in the airway and lung parenchyma compared to their C57BL/6 counterparts. We further demonstrated that the skewed IL-17/Th17 profile in C3H/HeN mice was predisposed by a higher basal level of IL-17 receptor C (IL-17RC) expression and then further amplified by a higher inducible IL-17RA expression in lungs. Most importantly, in vivo delivery of IL-17RA antagonist that resulted in a 50% reduction in the neutrophilic infiltration in lungs was able to reverse the susceptible phenotype of C3H/HeN mice to respiratory Chlamydia infection. Thus, our data for the first time have demonstrated a critical role for the IL-17/IL-17R axis in regulating host susceptibility to Chlamydia infection in mice.Chlamydia trachomatis is an obligate intracellular gram-negative bacterium that primarily infects epithelial cells lining the ocular, respiratory, and urogenital tract surfaces and causes many human diseases including trachoma, pneumonia, and pelvic inflammatory disease (2). Although effective antibiotics are available, the incidence of C. trachomatis infections continues to increase worldwide (41). In the United States alone, it is estimated that there are approximately 2.8 million new cases of urogenital C. trachomatis infection each year (58). An effective and safe Chlamydia vaccine is needed to address the global C. trachomatis epidemic, and a comprehensive understanding of the means of protective immunity and immunopathology of C. trachomatis infection is essential for vaccine development.C. trachomatis infection results in a wide variety of clinical manifestations, ranging from asymptomatic to mild or severe symptoms, acute or chronic inflammatory responses, and a wide range of chronic complications (5, 47, 59). Host genetic factors appear to be important in determining the outcome of Chlamydia infections. It has been reported that the increased incidence of Chlamydia-induced chronic diseases, such as tubal infertility and scarring trachoma, is correlated with certain human leukocyte antigen (HLA) haplotypes and polymorphism of genes encoding interleukin-10 (IL-10), CD14, and tumor necrosis factor alpha (6, 7, 16, 25, 44, 54). However, how these specific genes are involved in shaping the specific immune responses during Chlamydia infection in humans remains unclear. As in humans, inbred mouse strains, such as C57BL/6 (H-2^b), BALB/c (H-2^d), C3H/HeN (H-2^k), and DBA/2J (H-2^d) mice respond to respiratory (1, 39, 40, 63), genital (9-12, 52), and intraperitoneal (i.p.) (36) Chlamydia infections differently. C57BL/6 mice are regarded as a resistant strain, whereas BALB/c, DBA/2, and C3H/HeN mice are reported as susceptible strains with higher mortality, more prolonged bacterial burden, more severe tissue inflammatory responses (such as neutrophil infiltration), and higher rates of infertility following Chlamydia infection. Thus, these inbred mouse strains have been used extensively for identification of specific host factors that regulate immune responses and immune mechanisms underlying the pathogenesis of Chlamydia infection.Based on animal models (5, 8, 38, 50) and human studies (20, 21), T-cell-mediated immunity is essential in host defense against Chlamydia infection. Anti-Chlamydia immunity requires induction of potent Th1 cellular immunity that is characterized by production of IL-12 and gamma interferon (IFN-γ) (5, 37, 61). Mice deficient in IL-12 (17), IFN-γ (17, 23), or IFN-γ receptors (23, 24), or mice treated with anti-IL-12 or anti-IFN-γ antibody all demonstrated a marked inability to control Chlamydia infection, highlighting an unequivocal role of Th1 cellular immune response in host defense against Chlamydia infection. In contrast, skewed induction of Th2 immunity or a high level of IL-10 production increases susceptibility to Chlamydia infection in BALB/c mice by inhibiting IFN-γ production (9, 62, 63). While the theory of Th1/Th2 immune regulation has significantly advanced our understanding about anti-Chlamydia immunity, it does not appear to explain all genetically determined susceptibilities to Chlamydia infection. Susceptible C3H/HeN mice are able to mount comparable, if not higher, levels of Th1 immunity, or a similar ratio of Th1/Th2 responses as seen in resistant C57BL/6 mice (9, 39, 40). Thus, it is likely that other immune mechanisms are also involved in regulating differential susceptibilities to Chlamydia infection in different hosts.In the past few years, significant advances have been made in our understanding of T-cell immunity with the discovery of a unique αβ⁺ CD4⁺ T helper lineage termed Th17 cells (13, 19) that differ from the classic Th1 and Th2 lineages. Th17 cells produce novel proinflammatory cytokines including IL-17 (also called IL-17A) and IL-17F—two members of the IL-17 cytokine family, which has a total of six family members (IL-17A to IL-17F) and five receptors (IL-17 receptor A [IL-17RA] to IL-17RE) (27). In mice, Th17 lineage differentiation requires transforming growth factor beta and IL-6 for initiation and IL-23 for further expanding and becoming an established population (3, 30, 31, 35, 53). Although IL-17-producing CD4⁺ T cells represent one lineage of adaptive immunity, the IL-17/Th17 response functions as a classic innate immune component. Specifically, IL-17 and IL-17F interact with IL-17R, consisting of IL-17RA and IL-17RC subunits, to induce production of other proinflammatory cytokines (e.g., IL-1 and tumor necrosis factor), chemokines (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], CXCL2, and chemokine [C-C motif] ligand 2 [CCL2]) and growth factors (e.g., IL-6, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor) from tissue structural cells including fibroblasts and epithelial and endothelial cells. As a result, IL-17/Th17 response leads to an accumulation of neutrophils at the sites of infection and inflammation (27, 28). In addition to Th17 cells, γδ⁺ T cells and neutrophils are also capable of producing IL-17 in response to infectious agents including Mycobacterium tuberculosis (34), Escherichia coli (49), Aspergillus fumigatus (43), and Listeria monocytogenes (18). While a growing body of evidence indicates that IL-17-mediated neutrophilic response plays a critical role in host defense against extracellular bacteria (65, 66), IL-17-mediated responses are also responsible for severe tissue damage in other infection models (15, 45, 46), due largely to their potent effect on neutrophils. To date, however, the specific contribution of the IL-17/Th17 response in host susceptibility to Chlamydia infection remains unclear.In the present study, we investigated the role of the IL-17/Th17 response in host resistance against respiratory infection caused by the mouse pneumonitis biovar of Chlamydia muridarum in resistant C57BL/6 and susceptible C3H/HeN mice. The objectives of our study were (i) to characterize the IL-17/Th17 response in conjunction with well-characterized Th1 immunity, clinical evaluation, and bacterial clearance during C. muridarum infection; (ii) to investigate whether the IL-17/Th17 immune profile had any correlation with Chlamydia resistance and/or Chlamydia susceptibility, and (iii) if this does occur, to determine the mechanism(s) by which the IL-17/Th17 response modifies host immune responses to C. muridarum infection.     

The Caspase 1 Inflammasome Is Not Required for Control of Murine Lyme Borreliosis

The contribution of the inflammasome to the development of immune responses and disease during infection with the Lyme disease spirochete, Borrelia burgdorferi, is not well defined. Host defense against the spirochete is severely impaired in mice deficient in the adaptor molecule myeloid differentiation antigen 88 (MyD88), which is required not only for Toll-like receptor-mediated responses but also for the production of the proforms of interleukin 1β (IL-1β) and IL-18. These cytokines are released in active forms after cleavage by the inflammasome-associated enzyme caspase 1. To investigate the contribution of the inflammasome to host defense against B. burgdorferi, we examined Lyme borreliosis in mice deficient in either caspase 1 or apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), a molecule upstream of caspase 1 in the inflammasome signaling cascade. We found that caspase 1-deficient mice had a mild transient elevation in pathogen burden and a trend toward an increase in the prevalence of arthritis early in infection, but these differences resolved by day 14 postinfection. Caspase 1 deficiency had no effect on B. burgdorferi-induced humoral immunity, T-cell responses, or the abilities of macrophages to ingest and degrade spirochetes. The absence of the ASC protein had no effect on the control of the spirochete or the development of immune responses and disease. These findings reveal that the caspase 1 inflammasome is not critical to host defense against the extracellular pathogen Borrelia burgdorferi.Infection of humans with the Lyme disease spirochete, Borrelia burgdorferi, results in a characteristic pattern of skin lesions, arthritis, carditis, and neurologic abnormalities that reflect the immune response to the spirochete as it invades and disseminates in the mammalian host (7). In the murine model of Lyme borreliosis, spirochetes inoculated into the skin disseminate within days to infect all organ systems, but disease is primarily manifested in the joints and heart (4). Disease in the animal model is due largely to the innate immune response to spirochetes because histopathology reveals mainly neutrophils and macrophages within inflamed joints and hearts, respectively (5, 28, 36, 43), and occurs in the absence of adaptive (T- and B-cell-mediated) immunity (8, 28, 43).Recent studies have further defined the role of innate immunity in Lyme borreliosis. B. burgdorferi lipoproteins activate innate immune cells through the pattern recognition molecule Toll-like receptor 2 (TLR2), which is required for innate but not adaptive immune responses to the spirochete (2, 19, 49). Spirochete components also stimulate murine cells through TLR5 and TLR9 (44). The TLR cytosolic domains contain a Toll/interleukin 1 (IL-1) receptor domain (TIR) that interacts with myeloid differentiation antigen 88 (MyD88) and results in the activation of NF-κB and the production of proinflammatory cytokines, chemokines, and costimulatory molecules that are important for host defense (6, 12, 14). We and others have previously shown that B. burgdorferi-infected MyD88-deficient (MyD88^−/−) mice have significantly elevated pathogen burdens that persist through 90 days of infection despite the presence of high titers of anti-B. burgdorferi antibodies (9, 25). The elevated level of pathogen DNA in tissues was explained in part by our finding that MyD88^−/− peritoneal macrophages ingested spirochetes at the same rate as wild-type (WT) cells, but the kinetics of degradation was slower, with internalized spirochetes remaining in an elongated form for a longer period. Others have found that bone marrow-derived MyD88^−/− macrophages do not efficiently ingest spirochetes (44). The MyD88^−/− mice developed carditis and arthritis similar to the disease in WT mice analyzed at its peak (days 14 and 28) and during regression (day 45) (9, 25). Together, these results showed that MyD88-dependent signaling pathways are not required for B. burgdorferi-induced inflammation or disease regression but are necessary for efficient control of the pathogen burden by phagocytes. These studies did not distinguish whether interruption of MyD88-dependent TLR signaling pathways was solely responsible for the impaired control of the pathogen or whether other MyD88-dependent pathways also play a role.In addition to being a crucial signaling molecule for TLRs involved in B. burgdorferi recognition, MyD88 is required for IL-1 receptor (IL-1R)- and IL-18R-associated kinase signaling. TLR activation is a key inducer of the proforms of IL-1β and IL-18, and the secreted forms of these two cytokines require MyD88 for their receptors to mediate their effects (1, 34, 38). Behera et al. (6) have shown that IL-18 alone does not significantly contribute to host immunity in Lyme borreliosis because IL-18^−/− mice exhibit no defects in pathogen clearance or the development of disease. IL-1β, however, may play a role because human peripheral blood mononuclear cells secrete IL-1β after ingestion of live B. burgdorferi spirochetes (15). In support of this hypothesis, serum levels of IL-1β were reported to be elevated in Lyme disease patients, and the levels decreased significantly after doxycycline treatment (35). IL-1β mRNA levels in erythema migrans lesions were also shown to be elevated (31).To further delineate the role of MyD88-dependent signaling pathways in host defense against B. burgdorferi, we examined the course of Lyme borreliosis in mice deficient in either the intracellular cysteine protease caspase 1 or apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). Caspase 1 plays a key role in inflammatory responses by cleaving pro-IL-1β and pro-IL-18 into their active secreted forms (16, 22). These cytokines are matured in a large caspase 1-containing protein complex called the inflammasome (37). ASC, a component of the inflammasome, is required for eliciting the enzymatic activity of caspase 1. Caspase 1 contains an N-terminal caspase recruitment domain (CARD) shown to be involved in the assembly of protein platforms that promote proteolytic activation of recruited caspases in the context of apoptosis and inflammation (14). In addition to cleaving pro-IL-1 and pro-IL-18, caspase 1 is also involved in other proinflammatory pathways, including NF-κB signaling pathways associated with innate and adaptive immune responses (21, 23, 41). In contrast, ASC is essential only for the secretion of IL-1β/IL-18 but dispensable for caspase 1-mediated IL-6 and tumor necrosis factor alpha secretion and NF-κB and p38 activation (40). Thus, although both caspase 1^−/− mice and ASC^−/− mice have defects in the production of IL-1β/IL-18, caspase 1^−/− mice have additional defects in the activation of NF-κB.Several published reports have established that the inflammasome is important for immunity to intracellular bacteria and viruses, but much less is known about the contribution of the inflammasome to host defense against extracellular pathogens that elicit cytokines activated by caspase 1 (27, 29, 30, 32, 38, 42, 48). Thus, we sought to determine whether the inflammasome is also important during infection with the B. burgdorferi spirochete as representative of a subset of extracellular pathogens. We found that while B. burgdorferi can elicit IL-1β in a caspase 1-dependent fashion from mouse macrophages in vitro, the caspase 1-dependent inflammasome is not essential for the ultimate control of B. burgdorferi infection and disease.     

The K5 Capsule of Escherichia coli Strain Nissle 1917 Is Important in Mediating Interactions with Intestinal Epithelial Cells and Chemokine Induction

Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. coli. In this paper, we investigate the role of the K5 capsule in mediating interactions between Nissle 1917 and intestinal epithelial cells. We show that the loss of capsule significantly reduced the level of monocyte chemoattractant protein 1 (MCP-1), RANTES, macrophage inflammatory protein 2α (MIP-2α), MIP-2β, interleukin-8, and gamma interferon-inducible protein 10 induction by Nissle 1917 in both Caco-2 cells and MCP-1 induction in ex vivo mouse small intestine. The complementation of the capsule-minus mutation confirmed that the effects on chemokine induction were capsule specific. The addition of purified K5, but not K1, capsular polysaccharide to the capsule-minus Nissle 1917 at least in part restored chemokine induction to wild-type levels. The purified K5 capsular polysaccharide alone was unable to stimulate chemokine production, indicating that the K5 polysaccharide was acting to mediate interactions between Nissle 1917 and intestinal epithelial cells. The induction of chemokine by Nissle 1917 was generated predominantly by interaction with the basolateral surface of Caco-2 cells, suggesting that Nissle 1917 will be most effective in inducing chemokine expression where the epithelial barrier is disrupted.A probiotic has been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (20). These benefits include the balancing and restoration of the intestinal microflora, repair of intestinal barrier functions (54), expression of bacteriocins (36), immunomodulatory effects (18, 43, 47, 53), and antagonizing epithelial colonization and invasion by pathogens (2). Escherichia coli strain Nissle 1917 was isolated from the feces of a soldier who did not develop diarrhea during a severe outbreak of shigellosis (38). Despite exhibiting a serotype (O6:K5:H1) that is characteristic of E. coli strains associated with urinary tract infections, Nissle 1917 apparently is nonpathogenic (25, 53) and has been used widely in preventing infectious diarrheal diseases (7, 14, 27, 37, 52, 53), the treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn''s disease (7, 23, 32, 33), and to prevent the colonization of the digestive tract of neonates by pathogens (35). Recently, there has been a growing interest in investigating the immunomodulatory effect of Nissle 1917. Previous studies showed that colonization by Nissle 1917 may lead to an alteration of the hosts'' cytokine repertoire (13, 49), increased immunoglobulin A secretion (14), lymphocyte or macrophage activation (13), the modulation of CD4⁺ clonal expansion (47), the stimulation of antimicrobial peptide production by intestinal epithelial cells (39, 52, 54), and alterations of the pro- and anti-inflammatory balance of local cytokines (49). Recently it has been shown that Nissle 1917 activates γδT cells, stimulating CXCL8 and interleukin-6 (IL-6) release but inhibiting tumor necrosis factor alpha (TNF-α) secretion (26). Following activation, Nissle 1917 induced apoptosis in activated γδT cells, indicating a key role for Nissle 1917 in interacting with the subset of T cells that operate at the interface between the adaptive and innate immune responses (26). Nissle 1917 also has been shown to express a direct anti-inflammatory activity on epithelial cells by blocking TNF-α-induced IL-8 secretion through a NF-κB-independent mechanism (28). Although the immunomodulatory effects of Nissle 1917 are well documented, the contribution of individual microbial components in mediating such effects is less well understood. So far, only a role for flagellin in mediating the induction of human β-defensin expression by Nissle 1917 has been established (44). Nissle 1917 expresses a K5 capsule on its cell surface, and a number of roles for polysaccharide capsules in the virulence of E. coli have been proposed, including resistance to phagocytosis and complement-mediated killing and the increased colonization of the host (42). In contrast, in the case of other encapsulated pathogens, it has been shown that the expression of a polysaccharide capsule can affect the induction of chemokines following attachment to host cells (6, 17, 22, 24, 40, 41, 45, 50). The aim of the present study was to investigate the role of the K5 capsule in mediating the immunomodulatory activity of Nissle 1917.     

Association of Single Nucleotide Polymorphisms in Cytotoxic T-Lymphocyte Antigen 4 and Susceptibility to Autoimmune Type 1 Diabetes in Tunisians

In addition to HLA and insulin genes, the costimulatory molecule CTLA-4 gene is a confirmed type 1 diabetes (T1D) susceptibility gene. Previous studies investigated the association of CTLA-4 genetic variants with the risk of T1D, but with inconclusive findings. Here, we tested the contributions of common CTLA-4 gene variants to T1D susceptibility in Tunisian patients and control subjects. The study subjects comprised 228 T1D patients (47.8% females) and 193 unrelated healthy controls (45.6% females). Genotyping for CTLA-4 CT60A/G (rs3087243), +49A/G (rs231775), and −318C/T (rs5742909) was performed by PCR-restriction fragment length polymorphism (RFLP) analysis. The minor-allele frequencies (MAF) for the three CTLA-4 variants were significantly higher in T1D patients, and significantly higher frequencies of homozygous +49G/G and homozygous CT60G/G genotypes were seen in patients, which was confirmed by univariate regression analysis (taking the homozygous wild type as a reference). Of the eight possible three-locus CTLA-4 haplotypes (+49A/G, −318C/T, and CT60A/G) identified, multivariate regression analysis confirmed the positive association of ACG (odds ratio [OR], 1.93; 95% confidence interval [CI], 1.26 to 2.94), GCG (OR, 2.40; 95% CI, 1.11 to 5.21), and GTA (OR, 4.67; 95% CI, 1.52 to 14.39) haplotypes with T1D, after confounding variables were adjusted for. Our results indicate that CTLA-4 gene variants are associated with increased T1D susceptibility in Tunisian patients, further supporting a central role for altered T-cell costimulation in T1D pathogenesis.Type 1 (insulin-dependent) diabetes (T1D) is the most prevalent form of diabetes in children and young adults and results from autoimmune CD4⁺ and CD8⁺ T-cell-directed destruction of insulin-producing pancreatic β islet cells in genetically susceptible individuals (3, 12), leading to irreversible hyperglycemia and related complications (13). There is a strong genetic component to T1D pathogenesis, evidenced by its clustering in families and by the contributions of a number of susceptibility gene variants to its pathogenesis (10, 12, 29). They include the human leukocyte antigen (HLA) locus, in particular the class II region (DR and DQ), which accounts for 40 to 50% of T1D familial clustering (1, 12, 18), and non-HLA susceptibility loci, several of which were mapped by genome-scanning (11, 29) and/or candidate gene (7, 18, 31) approaches. They include insulin promoter gene variants, which reportedly may modulate immunological tolerance by controlling the expansion of the autoreactive cell pool (26), and the T-cell costimulator cytotoxic T-lymphocyte antigen 4 (CTLA-4) transmembrane glycoprotein, which plays a key role in the fine tuning of T-cell immunity (9, 32, 33).CTLA-4 is a 40-kDa transmembrane glycoprotein expressed on resting and activated T cells and nonlymphoid cells (33), and along with the related CD28 costimulatory molecule, it regulates T-cell activation (and is itself primarily mediated by engagement of the T-cell receptor [TCR]) but does recognize major histocompatibility complex (MHC)-bound antigenic peptides (9, 33). CTLA-4 negatively regulates T-cell activation and effector function, in part by inhibiting Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ]) cytokine production and IL-2 receptor α-chain (p55; Tac) expression by engaging antigen-presenting cell (APC)-bound B7.1 (CD80) and B7.2 (CD86) ligands (9, 33). Functionally, CTLA-4 attenuates T-cell signaling by interference with intracellular signal transduction events, including TCR signaling, and reduced CTLA-4 expression and/or activity results in uncontrolled T-cell-associated autoimmunity and lymphoproliferative disease (9, 21). In this regard, it was shown that CTLA-4 polymorphisms significantly influence the risk of autoimmune diseases, including Graves'' disease, systemic lupus erythematosus, autoimmune hypothyroidism, celiac disease, and type 1 diabetes (15, 21, 32).First observed in Italian subjects (25), and confirmed subsequently by case control and family studies, CTLA-4 polymorphic variants were linked with T1D pathogenesis (14, 20, 31, 32). While this association was detected in different ethnic groups (14, 23, 30), it appears more likely to be Caucasian selective (10, 29, 33) and absent from non-Caucasians (5, 6, 8, 19, 22). A recent report from the Type I Diabetes Genetics Consortium bearing on 2,300 affected sib pair families demonstrated that among the 24 single nucleotide polymorphisms (SNPs) genotyped in the CTLA-4 region, only the +49A/G and CT60 SNPs were replicated in the nine combined collections (27). In the present study, we investigated the association of three common CTLA-4 SNPs (−318C/T; +49A/G, and CT60A/G) and the corresponding haplotypes with T1D in Tunisian Arab patients.     

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.     

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.     

Toxoplasma gondii Cyclophilin 18-Mediated Production of Nitric Oxide Induces Bradyzoite Conversion in a CCR5-Dependent Manner

Toxoplasma gondii modulates pro- and anti-inflammatory responses to regulate parasite multiplication and host survival. Pressure from the immune response causes the conversion of tachyzoites into slowly dividing bradyzoites. The regulatory mechanisms involved in this switch are poorly understood. The aim of this study was to investigate the immunomodulatory role of T. gondii cyclophilin 18 (TgCyp18) in macrophages and the consequences of the cellular responses on the conversion machinery. Recombinant TgCyp18 induced the production of nitric oxide (NO), interleukin-12 (IL-12), and tumor necrosis factor alpha through its binding with cysteine-cysteine chemokine receptor 5 (CCR5) and the production of gamma interferon and IL-6 in a CCR5-independent manner. Interestingly, the treatment of macrophages with TgCyp18 resulted in the inhibition of parasite growth and an enhancement of the conversion into bradyzoites via NO in a CCR5-dependent manner. In conclusion, T. gondii possesses sophisticated mechanisms to manipulate host cell responses in a TgCyp18-mediated process.Toxoplasma gondii is a ubiquitous protozoan parasite that is able to infect a broad range of warm-blooded animals, including humans (17, 38). Fortunately, T. gondii is a well-adapted parasite which generally causes very little disease unless the host''s immune system is compromised in situations such as AIDS (41). Toxoplasma gondii affects pro- and anti-inflammatory host cell signaling in such a way as to maximize parasite multiplication and spread while maintaining host survival (14). One aspect of this manipulation is the upregulation of the interleukin-12 (IL-12)-dependent production of gamma interferon (IFN-γ), which is critical to host survival of acute toxoplasmosis (7, 8, 26, 28). This effect appears to occur by a pathway unique to T. gondii and involves the triggering of cysteine-cysteine chemokine receptor 5 (CCR5) in dendritic cells (DC) and macrophages by secreted T. gondii cyclophilin 18 (TgCyp18) (2). High et al. previously isolated genes encoding two Toxoplasma gondii cyclophilins, TgCyp18 and TgCyp20 (27). In T. gondii, the isolation of cyclosporine-binding proteins on affinity columns yielded only the cyclophilins TgCyp18 and TgCyp20 (27). Both cyclophilins were highly similar to human cyclophilin (hCyp18) in the central core region, but TgCyp20 differed in a 7-amino-acid “insertion” in the same region as that in Plasmodium falciparum cyclophilins (27).TgCyp18, but not hCyp18 or P. falciparum cyclophilin 19A (PfCyp19A), appears to induce IL-12 production by interacting directly with CCR5, an effect that was blocked by the addition of cyclosporine (2, 4, 63). These observations implied that structural determinants of TgCyp18, related to cyclosporine binding, were responsible for the induction of IL-12 synthesis (4, 63). This idea was confirmed by modeling of the TgCyp18 structure on that of PfCyp19A and site-directed mutagenesis of putatively surface-exposed residues that were absent in PfCyp19A (63). Two of the TgCyp18 mutants, namely, ¹⁷GEH¹⁹ to ¹⁷AAA¹⁹ and ¹⁴⁹RP¹⁵⁰ to ¹⁴⁹YV¹⁵⁰, located in the N and C termini of the protein, respectively, had reduced interactions with CCR5 and reduced IL-12 induction (63). Moreover, TgCyp18 peptidyl-prolyl cis-trans isomerase (PPIase) activity was not required for its interaction with CCR5, but IL-12 induction by TgCyp18 required both CCR5 binding and PPIase enzymatic activities (63). TgCyp18 appears to act as a structural mimic of CCR5-binding ligands, albeit one with no sequence similarity to the known host ligands, macrophage inflammatory protein 1α/chemokine (C-C motif) ligand 3 (CCL3), macrophage inflammatory protein 1β/CCL4, regulated on activation normal T-cell expressed and secreted (RANTES)/CCL5, or monocyte chemotactic protein 2/CCL8, for this receptor (4, 63). There is also evidence that the closely related protozoan Neospora caninum cyclophilin plays a role in stimulating IFN-γ production by bovine peripheral blood mononuclear cells and N. caninum-specific CD4⁺ T cells (59). This effect is also blocked by cyclosporine (59). IFN-γ production induced by N. caninum tachyzoites is thought to be critical in controlling the acute phase of neosporosis (59).Pressure from the immune response causes tachyzoites to differentiate into slowly multiplying bradyzoites, which form cysts within muscle and brain cells (19). The cysts are protected from the host immune response and establish a life-long chronic infection (18). If the tissue cysts are ingested, for example, through the consumption of undercooked meat, bradyzoites are released into the gut, invade epithelial cells, and differentiate into tachyzoites, initiating a new asexual cycle (10). In vitro models of tachyzoite-to-bradyzoite differentiation have been established by using a variety of stress conditions that mimic the stresses of the host immune response. These conditions include treatment with IFN-γ (5), mitochondrial inhibitors (6), alkaline pH (pH 8.1) (53), and high temperature (54). The stress response is controlled in part by eukaryotic initiation factor 2 kinase in the parasite, which is well characterized as a stress response in eukaryotic cells (55), and differentiation also involves parasite-derived cyclic nucleotide kinases (20). Large-scale sequencing of stage-specific cDNA (36, 39), microarray studies (10), and serial analysis of gene expression tags (44) revealed that stage conversion involves changes in the levels of expression of a large number of genes, although the regulatory mechanism(s) involved in the conversion is poorly understood.Although previous reports mentioned the ability of TgCyp18 to induce the production of the IL-12 (1, 2, 14, 63), the productions of other cytokines were not elucidated. Here we show that TgCyp18 induces the production of other cytokines and nitric oxide (NO) and enhances the bradyzoite conversion of T. gondii.     

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.