CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28^−/−) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28^−/− mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro- and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.It has long been appreciated that cellular immunity is the most important resistance mechanism against fungal infections (14, 36, 64). CD4⁺ and CD8⁺ T-cell subpopulations have been described to have a fundamental role in the control of fungal growth, and disease severity is also controlled by regulatory T (Treg) cells, which prevent tissue pathology by controlling excessive inflammatory reactions (25, 45, 46, 65). Similar to other deep mycoses, the severity of paracoccidioidomycosis (PCM), the most severe pulmonary mycosis in Latin America, is controlled by cellular immunity and cytokine-activated phagocytes that are able to kill Paracoccidioides brasiliensis, the etiological agent of this infection (10, 20, 30, 60, 61). In humans and in murine models of PCM, resistance to the disease is associated with the secretion of gamma interferon (IFN-γ) and other type 1 cytokines, whereas impaired Th1 immunity and the prevalent secretion of Th2 cytokines correlate with a systemic and progressive disease (2, 6, 39, 59, 76). Studies with CD4⁺ and CD8⁺ T-cell-deficient mice revealed that both T-cell subsets are involved in the protective immunity against P. brasiliensis infection and indicated the prominent role of CD8⁺ T cells (3, 21, 25). Besides the prevalent Th2 immunity, recent investigations have described alternative mechanisms underlying T-cell dysfunction in humans and experimental PCM. Increased apoptosis and overexpression of Fas and FasL in T cells suggest that activation-induced cell death (AICD) is a mechanism that controls T-cell expansion during the active disease (13, 19). In addition, the increased expression of CTLA-4 and the expansion of Treg cells were associated with severe patterns of the disease (24, 45, 46, 56). Thus, in addition to cytokine imbalance, other regulatory mechanisms can actively participate in the unresponsiveness of T cells in P. brasiliensis-infected hosts.Optimal activation, proliferation, and cytokine production by antigen-specific T cells require two distinct signals from dendritic cells or other antigen-presenting cells (APCs). After T-cell receptor (TCR) occupancy by the antigen epitope/major histocompatibility (MHC) complex (first signal), a second signal is mediated by costimulatory molecules (43, 63), such as CD28 on T cells and their counter-receptors CD80 (B7-1) and CD86 (B7-2) expressed by APCs (1, 34). Soluble molecules, such as cytokines and chemokines, also participate in the activation process, which drives and controls T-cell numbers and fates (1). CD28 enhances the TCR-triggered activation of naïve T cells, promotes interleukin-2 (IL-2) secretion and prevents T-cell anergy (1, 37). Alternatively, CD28-independent T-cell activation can occur if a strong and sustained antigen-specific signal is available (40, 81). Like CD28, two other molecules, cytotoxic T-lymphocyte antigen-4 (CTLA-4) and mouse inducible costimulatory molecule (ICOS), are selectively expressed by T cells, but the expression of these molecules depends on previous cell activation (50, 71). More recently, evidence has emerged that CD28 family members are also crucial regulators of natural and induced regulatory (CD4⁺CD25⁺Foxp3⁺) T cells (9). These cells are induced in the thymus and in the periphery, respectively, and control self-tolerance and the activation of several components of innate and adaptive immunity (68). Treg cells can suppress immune responses through the production of immunosuppressive cytokines (mainly IL-10 and transforming growth factor β [TGF-β]), through the induction of the apoptosis of effector T cells and through the modification of the functional properties of antigen-presenting cells (70, 78).Immunoprotection against microorganisms has been shown to be either CD28 dependent or independent. CD28-deficient (CD28^−/−) mice are highly susceptible to infection with Salmonella enterica serovar Typhimurium due to the poor ability of these mice to secrete IFN-γ (51). During some viral and parasitic infections, CD28 was shown to be required to mediate CD8⁺ T-cell immunoprotection (8, 53). In contrast, CD28^−/− mice infected with Mycobacterium bovis or Listeria monocytogenes control the bacterial burden and develop cell-mediated immunity (35, 52). In primary and opportunistic fungal infections, CD28 costimulation controls protective immunity, the expansion and function of regulatory T cells, and the intensity of inflammatory reactions (5, 54, 55, 66, 84).Because CD28 is critical for T-cell activation in fungal infections, we investigated its role in a murine model of P. brasiliensis infection. We show that CD28 costimulation exerts contrasting roles in pulmonary PCM. Early in infection, CD28 expression results in efficient adaptive immunity that is able to control fungal growth. Late in infection, however, this costimulatory molecule induces significant expansion of regulatory T cells, diminished immunity, and uncontrolled fungal growth that eventually leads to the death of the mice. In contrast, the absence of CD28 costimulation results in impaired T-cell immunity, which appeared to be compensated by the absence of Treg cell expansion. This weak but persistent immunity was able to partially control fungal growth, organize granulomatous lesions, and guarantee the enhanced survival of the mice, suggesting the relative protection conferred by CD28-independent mechanisms.     

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

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

Development of a secondary immune response to Mycobacterium tuberculosis is independent of Toll-like receptor 2

Published work indicates that the contribution of Toll-like receptor 2 (TLR2) to host resistance during acute Mycobacterium tuberculosis infection is marginal. However, in these studies, TLR2 participation in the memory immune response to M. tuberculosis was not determined. The substantial in vitro evidence that M. tuberculosis strongly triggers TLR2 on dendritic cells and macrophages to bring about either activation or inhibition of antigen-presenting cell (APC) functions, along with accumulating evidence that memory T cell development can be calibrated by TLR signals, led us to question the role of TLR2 in host resistance to secondary challenge with M. tuberculosis. To address this question, a memory immunity model was employed, and the response of TLR2-deficient (TLR2 knockout [TLR2KO]) mice following a secondary exposure to M. tuberculosis was compared to that of wild-type (WT) mice based on assessment of the bacterial burden, recall response, phenotype of recruited T cells, and granulomatous response. We found that upon rechallenge with M. tuberculosis, both WT and TLR2KO immune mice displayed similarly enhanced resistance to infection in comparison to their naïve counterparts. The frequencies of M. tuberculosis-specific gamma interferon (IFN-γ)-producing T cells, the phenotypes of recruited T cells, and the granulomatous responses were also similar between WT and TLR2KO immune mice. Together, the findings from this study indicate that TLR2 signaling does not influence memory immunity to M. tuberculosis.Mycobacterium tuberculosis expresses a large repertoire of lipoproteins that can trigger signaling from Toll-like receptor 2 (TLR2) (13), including the 19-kDa lipoprotein (LpqH) (5), LprA (Rv1270) (29), and LprG (Rv1411c) (11). In addition to lipoproteins, lipomannan (31) and phosphatidyl-myo-inositol mannoside (PIM) (12, 16) also interact with TLR2 to initiate cellular activation (16). Despite this collection of TLR2 agonists on the tuberculosis (TB) bacillus, murine studies indicate that TLR2 is not essential for host resistance against acute M. tuberculosis infection (34, 37).It is well appreciated that memory immunity in tuberculosis does not provide long-term protective immunity, as evidenced in humans and experimental infections of mice. In a study performed in Cape Town, South Africa, it was determined that the incidence rate of TB attributable to reinfection after successful chemotherapy was four times that of new TB (40). In mouse models, immunological memory induced by M. tuberculosis infection can provide short-term protection, as evidenced by early reduction in the bacterial burden in the lungs following reexposure (6, 36). The memory immune mice exhibit a transient early induction of Th1 cells compared to naïve mice and concomitant early control of bacterial replication. However, despite the skewed kinetics, the memory mice do not achieve bacterial sterility in the lung, and bacteria continue to be maintained in a stable state. Clearly, this major gap in our understanding of how to induce sterilizing memory immunity in TB is an impediment to vaccine development.In vitro studies have documented opposing outcomes from antigen-presenting cells (APC) following interaction of their surface TLR2 with M. tuberculosis. For example, TLR2 signals upregulate B7 expression, induce interleukin 12 (IL-12) secretion (15), and initiate antimicrobial responses within M. tuberculosis-infected macrophages (22). TLR2 also initiates signaling that inhibits major histocompatibility complex (MHC) class II-dependent antigen presentation (24, 26) by macrophages and responsiveness to gamma interferon (IFN-γ) (2, 8, 18, 27). How these opposing changes to APC by TLR2 signals affect naïve T cell differentiation into effector and memory T cells following M. tuberculosis infection remains unclear. Furthermore, recent studies indicate that high expression levels of IL-12 in the environment promote effector T cell development while low expression levels (17) or even the absence (28, 41) of the cytokine is favorable for central memory T cell development. Together with the report that TLR2 regulates IL-10 production from macrophages and dendritic cells (DC) following M. tuberculosis infection (15, 30), these findings suggest that TLR2 signals may modulate the inflammatory milieu during T cell priming to influence effector versus memory T cell development.The literature on inhibition of APC function by TLR2 predicts that removal of TLR2 may improve APC functions and lead to better memory immunity. On the other hand, the finding that TLR2 signaling is anti-inflammatory and consequently conducive to memory T cell development predicts that absence of TLR2 may result in poorer memory immunity. Therefore, in this study, we examined whether the absence of TLR2 improved or worsened the capacity of the host to generate memory immunity upon rechallenge with M. tuberculosis.     

Mucosal Administration of Flagellin Protects Mice from Streptococcus pneumoniae Lung Infection

Streptococcus pneumoniae is a major cause of pneumonia in infants and the elderly. Innate defenses are essential to the control of pneumococcal infections, and deficient responses can trigger disease in susceptible individuals. Here we showed that flagellin can locally activate innate immunity and thereby increase the resistance to acute pneumonia. Flagellin mucosal treatment improved S. pneumoniae clearance in the lungs and promoted increased survival of infection. In addition, lung architecture was fully restored after the treatment of infected mice, indicating that flagellin allows the reestablishment of steady-state conditions. Using a flagellin mutant that is unable to signal through Toll-like receptor 5 (TLR5), we established that TLR5 signaling is essential for protection. In the respiratory tract, flagellin induced neutrophil infiltration into airways and upregulated the expression of genes coding for interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), CXCL1, CXCL2, and CCL20. Using depleting antibodies, we demonstrated that neutrophils are major effectors of protection. Further, we found that B- and T-cell-deficient SCID mice clear S. pneumoniae challenge to the same extent as immunocompetent animals, suggesting that these cell populations are not required for flagellin-induced protection. In conclusion, this study emphasizes that mucosal stimulation of innate immunity by a TLR not naturally engaged by S. pneumoniae can increase the potential to cure pneumococcal pneumonia.Streptococcus pneumoniae (pneumococcus) causes respiratory infections among infants and the elderly worldwide (40, 44). Capsular polysaccharide is the main virulence factor, and its composition defines 91 serotypes of pneumococcus (42). Certain serotypes colonize the human nasopharynx asymptomatically, representing a reservoir for interindividual transmission of the bacteria. In some individuals, colonization may progress to pneumococcal pneumonia and invasive disease (19, 36). In contrast, other serotypes, such as serotype 1, are rarely associated with colonization but cause invasive infections (28).Activation of innate defenses is essential for the control of pneumococcal infection (1, 20). Toll-like receptor 2 (TLR2), TLR4, and TLR9, as well as the adaptor MyD88, participate in the early detection and clearance of pneumococcus in the lungs (reviewed in reference 42). The cytosolic receptors Nod1 (nucleotide-binding oligomerization domain 1) and Nod2 are also involved in the recognition of pneumococci (29). TLR signaling activates mucosal innate responses that culminate with the recruitment of phagocytes, such as polymorphonuclear neutrophils (PMN) and macrophages, and the production of microbicidal agents (for a review, see reference 8). This process triggers rapid eradication of the pathogen by phagocytosis as well as by extracellular killing. In MyD88-deficient animals, S. pneumoniae is unable to intrinsically trigger any PMN recruitment into the airways, and animals thus have increased susceptibility to pneumonia (1). The contribution of TLR signaling in humans has been highlighted by a recent study showing that some MyD88 polymorphisms are associated with increased susceptibility to pneumococcal infection (43).The modulation of immunity by the activity of innate receptors to elicit protective responses against infections is an emerging concept (6, 35). The rationale is to promote innate responses that greatly exceed in magnitude, quality, and dynamics the innate response triggered by the pathogen itself. The effectiveness of TLR agonists for therapeutic treatment of infectious diseases has been demonstrated in several animal models, including models of respiratory infections (6, 21, 35). TLR5 senses bacterial flagellins, which are the main constituents of flagella. Various cells of the pulmonary tract, including the epithelial cells (14, 33), express TLR5, and mucosal administration of flagellin induces MyD88-dependent signaling, characterized by the swift production of various proinflammatory cytokines and chemokines (3, 10, 15, 16, 27, 33), as well as by rapid and heavy neutrophil infiltration into the airways (2, 10, 16). Although S. pneumoniae does not have flagella, we hypothesized that activation of TLR5 signaling may promote new and appropriate protective innate defenses against ongoing acute pneumococcal infections. Here we report that local stimulation of innate immunity by flagellin from Salmonella enterica serovar Typhimurium blocks the progression of pneumococcal pneumonia in mice.     

Regulation of innate immune response to Candida albicans infections by αMβ2-Pra1p interaction

Candida albicans is a common opportunistic fungal pathogen and is the leading cause of invasive fungal diseases in immunocompromised individuals. The induction of cell-mediated immunity to C. albicans is one of the main tasks of cells of the innate immune system, and in vitro evidence suggests that integrin α_Mβ₂ (CR3, Mac-1, and CD11b/CD18) is the principal leukocyte receptor involved in recognition of the fungus. Using α_Mβ₂-KO mice and mutated strains of C. albicans in two models of murine candidiasis, we demonstrate that neutrophils derived from mice deficient in α_Mβ₂ have a reduced ability to kill C. albicans and that the deficient mice themselves exhibit increased susceptibility to fungal infection. Disruption of the PRA1 gene of C. albicans, the primary ligand for α_Mβ₂, protects the fungus against leukocyte killing in vitro and in vivo, impedes the innate immune response to the infection, and increases fungal virulence and organ invasion in vivo. Thus, recognition of pH-regulated antigen 1 protein (Pra1p) by α_Mβ₂ plays a pivotal role in determining fungal virulence and host response and protection against C. albicans infection.Candida albicans is an opportunistic pathogen, a pleomorphic fungus existing in yeast or filamentous forms (19, 23). Although the yeast form can bind to gut mucosal membranes with further colonization (20, 49), it is thought that the filamentous morphology provides some advantage during interaction with the mammalian immune system as a part of fungal anti-host defense, and the ability of C. albicans to rapidly and reversibly switch between yeast and filamentous morphologies is crucial to its pathogenicity (16, 55, 65, 82). In recent years, Candida infections ranked as the fourth most common cause of bloodstream infections and are the leading cause of life-threatening nosocomial fungal infections (1, 87). The risk of developing opportunistic bloodstream infections is greatly increased in patients who are severely immunocompromised. Candida strains that are resistant to commonly used antimycotics have emerged rapidly (72, 98). Therefore, dissecting its pathogenic mechanisms and the host response to C. albicans infections is of great importance.The innate immune system provides the principle protection against disseminated candidiasis. Polymorphonuclear leukocytes (PMNs) have been shown to be the primary components of the host''s innate immune defenses against Candida infections (27, 59, 67). The most prominent receptors on leukocytes utilized in fungal or microbial recognition are the integrins of the beta-2 subfamily (62, 63). These cell surface receptors mediate migration of leukocytes to sites of infection and adhesion to microorganisms with subsequent phagocytosis and killing of many pathogens (12, 27, 62). Patients with defects in leukocyte phagocytic functions, such as individuals with the rare (that is, ca. 1 in 10⁶ people) hereditary disease, leukocyte adhesion deficiency 1 (LAD-1), which is characterized by the low expression (mild LAD-1) or complete absence (severe) of all four of the beta-2 integrins due to mutations in the β₂ gene (4, 5, 34), are highly susceptible to wide range of bacterial and fungal infections (7, 50, 80). Staphylococcus aureus and Streptococcus spp. are the most common bacterial pathogens, and Candida spp. are the primary fungi isolated from patients with LAD-1. The candidal skin infections described in older publications were reported to occur in ca. 16% of patients; Candida esophagitis has also been frequently reported (6, 7). In three more-recent reports, a total of 27 cases of LAD-1 patients were described, all of which had infections of different etiologies: C. albicans infections in 13 patients (48%), 17 patients (63%) dead from infections, and in 6 cases (22% of all LAD-1 patients or 46% of all cases of mortality) the cause of death was fungal septicemia (44, 65, 71). In most cases, the degree of severity of these symptoms may be correlated with the level of β₂ expression on the patients’ leukocytes. Generally, patients with <1% normal levels of β₂ are the most susceptible to frequent and life-threatening systemic infections and usually do not survive to adulthood. Patients with higher levels of expression (up to 10% of normal) develop milder forms of LAD-1 and may survive to adulthood with proper medical care (10, 86). The fungal invasion usually starts in newborns and toddlers (severe LAD-1) or in children (milder) as recurrent, severe skin and soft tissue infections that tend to be necrotic, leading to colitis, otitis, pneumonia with spontaneous peritonitis, and the formation of nodular and ulcerative lesions in later stages, which ultimately lead to sepsis and death (44, 53, 64). The principle beta-2 integrin involved in recognition of bacterial and fungal pathogens is α_Mβ₂ (Mac-1, CD11b/CD18, and CR3) (37, 39). α_Mβ₂ is a pivotal adhesion receptor on PMNs, cells of the monocytoid lineage, subsets of T lymphocytes, and NK cells (37, 38). The capacity of α_Mβ₂ to support leukocyte adhesion, migration, and phagocytosis depends upon its ability to recognize and mediate responses to a diverse set of structurally unrelated ligands, including fibrinogen (104), complement fragment iC3b (11), and intracellular cell adhesion molecule-1 (ICAM-1, CD54), as well as numerous bacterial lipoproteins and fungal mannans and β-glycans (101, 103). This cell surface receptor consists of two structurally distinct subunits, α_M and β₂, which associate noncovalently. The α_M subunit is unique to this receptor, and the β₂ subunit is shared with three other members of the beta-2 integrin subfamily: LFA-1 (α_Lβ₂), p150,95 (α_Xβ₂), and α_Dβ₂ (reviewed in references 43 and 52). Of the beta-2 integrins, α_Mβ₂ has been specifically implicated in the interaction of leukocytes with C. albicans: PMNs utilize α_Mβ₂ to adhere only to the filamentous form of C. albicans and not to yeast cells (36, 37, 39). Although other leukocyte receptors, Dectin-1 and Toll-like receptor 2 (TLR2), which can bind fungal β-glucan (13, 68, 99), and mannan-binding TLR4 (69) also participate in fungal recognition and are essential in leukocyte activation and eventually in activation of β-2 integrins (42, 91), they do not directly mediate leukocyte migration and adhesion. Also Dectin-1 and TLR2 recognize yeast forms of C. albicans only (41).Recently, we identified C. albicans pH-regulated antigen 1 protein (Pra1p) (84), also known as fibrinogen binding protein 1 (Fbp1) (56) or C. albicans 58-kDa mannoprotein (mp58) (17), as the major ligand of α_Mβ₂ among C. albicans proteins (88). Pra1p is expressed predominantly on the surface of the hyphal form and not the yeast form of C. albicans (25, 66, 85). The expression of Pra1p is strongly pH dependent, requiring a pH > 7, and is also regulated by nutrition and certain other fungal genes (25, 79, 84). It is a mannoprotein, with carbohydrate moieties accounting for 18 to 30% of its molecular mass (19, 25). In addition to fibrinogen, Pra1p also binds factor H, factor H-like protein, and plasminogen (58). Although small quantities of Pra1p may be present on the yeast form of C. albicans, it becomes highly glycosylated only on the filamentous forms (hyphal and pseudohyphal forms) (18, 19), and we found that sugar residues are important for recognition of C. albicans by α_Mβ₂ (39, 88). However, the biological role and the significance of the α_Mβ₂-Pra1p interaction in C. albicans virulence and host defense is unknown. The present study was undertaken to determine the role and significance of the α_Mβ₂-Pra1p interaction in fungal pathogenicity and its effects on host defense in vivo using α_Mβ₂-deficient knockout (KO) mice and the Δpra1 strain of C. albicans in two distinct models of acute murine candidiasis.     

Optimal CD8 T-Cell Response against Encephalitozoon cuniculi Is Mediated by Toll-Like Receptor 4 Upregulation by Dendritic Cells

CD8⁺ T-cell immunity has been shown to play an important role in the protective immune response against Encephalitozoon cuniculi. Although earlier studies suggest that dendritic cells (DC) are important for the induction of this response, the factors responsible for initiation of the dendritic cell response against this pathogen have not been evaluated. In the current study, we demonstrate that E. cuniculi infection causes strong Toll-like receptor 4 (TLR4)-dependent dendritic cell activation and a blockade of this molecule reduces the ability of DC to prime an antigen-specific CD8⁺ T-cell response. Pretreatment of DC with anti-TLR4 antibody causes a defect in both in vitro and in vivo CD8⁺ T-cell priming. These findings, for the first time, emphasize the contribution of TLR4 in the induction of CD8⁺ T-cell immunity against E. cuniculi infection.Microsporidia are small obligate intracellular parasites that, until recently, were thought to be protozoans; however, evidence now suggests that they are related to fungi (15, 17). Microsporidia can infect a vast number of species of vertebrates and invertebrates; of the 150 genera of microsporidia, however, only 7 have been found to infect humans (13). Severe infections have been reported predominantly for immunocompromised patients, such as patients with HIV and organ transplant recipients (2, 7, 23, 37). Acute infections have also been reported in travelers and the elderly (26, 27), and there is evidence of colonization of healthy, nonsymptomatic patients (34).Due to the prevalence of opportunistic microsporidian infections associated with the HIV-AIDS pandemic, recent research has focused on the host''s immune response to these pathogens. Early animal studies showed that cellular immunity was necessary to protect SCID mice from a lethal Encephalitozoon cuniculi challenge. Moreover, depletion of CD8⁺ T cells caused mice to succumb to intraperitoneal (i.p.) E. cuniculi infection (21), and previous studies in our laboratory have shown that cytotoxic lymphocytes play a major role in protection against this effect (20, 21).Recent reports from our laboratory have demonstrated that dendritic cells (DC) play an important role in the priming of the immune response against E. cuniculi (31, 32). T cells incubated with E. cuniculi-pulsed DC exhibited antigen-specific characteristics, specifically gamma interferon (IFN-γ) production, cytotoxicity, and proliferation (31, 32). In order to mount an immune response against a foreign pathogen, DC must first recognize the pathogen to initiate an appropriate response. One key method of recognition is through Toll-like receptors (TLRs), which were first discovered in Drosophila in response to infection with fungal pathogens (24). However, specific TLR molecules involved in DC activation during E. cuniculi infection have not been identified previously. We evaluated the upregulation of specific molecules involved in activation of the DC response after E. cuniculi infection. Different TLR molecules were tested, and TLR4 expression was found to be essential for induction of the optimal CD8⁺ T-cell response by these cells.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

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

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

Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage Functions

Cryptococcus neoformans and distantly related fungal species release extracellular vesicles that traverse the cell wall and contain a varied assortment of components, some of which have been associated with virulence. Previous studies have suggested that these extracellular vesicles are produced in vitro and during animal infection, but the role of vesicular secretion during the interaction of fungi with host cells remains unknown. In this report, we demonstrate by fluorescence microscopy that mammalian macrophages can incorporate extracellular vesicles produced by C. neoformans. Incubation of cryptococcal vesicles with murine macrophages resulted in increased levels of extracellular tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), and transforming growth factor β (TGF-β). Vesicle preparations also resulted in a dose-dependent stimulation of nitric oxide production by phagocytes, suggesting that vesicle components stimulate macrophages to produce antimicrobial compounds. Treated macrophages were more effective at killing C. neoformans yeast. Our results indicate that the extracellular vesicles of C. neoformans can stimulate macrophage function, apparently activating these phagocytic cells to enhance their antimicrobial activity. These results establish that cryptococcal vesicles are biologically active.Cryptococcus neoformans is an encapsulated yeast that causes disease in diverse species, including humans. Infection is most commonly acquired by inhalation of environmental propagules. C. neoformans rarely causes disease in immunocompetent individuals, but patients with immunological disorders can develop disseminated and neural cryptococcosis (63).Extracellular microbial products have been amply demonstrated to modulate the interaction between host cells and pathogens. Many virulence factors and immunogens are released in their soluble forms by fungal cells to the extracellular space (4, 9, 16, 19, 37, 49, 53, 60, 62, 65, 67). C. neoformans, for instance, constitutively secretes large amounts of its capsular polysaccharide glucuronoxylomannan (GXM) (61). Disease progress is associated with detection of GXM, which is a potent modulator of the immune response (reviewed in reference 81). Other secreted virulence-related factors include galactoxylomannan (GalXM) (14), phospholipases (16), and urease (12, 62). In addition to acting as virulence factors, culture supernatant components are immunogenic, conferring protection against C. neoformans infection (51, 53).Phagocytes are particularly important effector cells in the control of systemic mycoses (54). The interaction of C. neoformans with phagocytes, including macrophages, monocytes, dendritic cells, and neutrophils, has been widely studied (23, 32, 43, 46, 50, 59, 68, 77). Cryptococcal GXM is antiphagocytic (34) and a powerful immunomodulator (45, 79). C. neoformans capsule size directly correlates with the efficacy of phagocytosis in vitro (6, 15, 82). Phagocytosis of C. neoformans can result in either fungal killing (24, 30) or survival (2, 3, 39-41, 71, 80). Killing of C. neoformans apparently involves the production of oxidative species (24), while the mechanisms of fungal escape include phagosome extrusion, cell-to-cell spread, and phagosomal permeabilization (2, 3, 40, 41, 71). Capsular polysaccharides and melanin are known to modulate the interaction of C. neoformans with phagocytes in favor of the fungus (27, 39, 47, 48, 71, 72, 74, 76), but the role of other structures in the outcome of yeast phagocytosis is virtually unknown.A number of recent studies have shown that GXM, GalXM, pigments, proteins, and lipids are trafficked in vesicles that traverse the cell wall (7, 14, 20, 56, 57, 62, 64, 65). Extracellular vesicles are also produced by the pathogens Candida albicans, C. parapsilosis, Sporothrix schenckii, and Histoplasma capsulatum, as well as by the model yeast Saccharomyces cerevisiae (1), suggesting that extracellular vesicle secretion is a general property of fungal cells. Secreted vesicles are heterogeneous. For instance, vesicles secreted by C. neoformans were classified into four different groups based on morphology and electron density (64). Additionally, vesicle diameter ranges from 30 to 400 nm, with the majority having dimensions of 100 to 150 nm (20, 64, 65). The combined use of serology, biochemistry, proteomics, and lipidomics led to the identification of 2 polysaccharides, phospholipids, 4 neutral lipids, and 76 proteins as extracellular vesicle components secreted by C. neoformans, which means that at least 81 different molecules are released to the extracellular milieu by vesicular secretion (14, 57, 64). It is likely that this number is an underestimate resulting from the difficulty of proteomic studies in vesicles from highly encapsulated cryptococcal cells, since a higher number of vesicular proteins were characterized in other fungi. For example, in H. capsulatum, proteomics and lipidomics of extracellular vesicles revealed an even more complex composition, including 283 proteins and 17 different phospholipids (1).In this study, we evaluated the influence of extracellular vesicles on the fate of C. neoformans after phagocytosis by mouse macrophages. Our results show that fungal vesicles are biologically active and stimulate macrophages. Moreover, our results demonstrate that vesicles from an acapsular mutant strain were more effective in eliciting macrophage activation and augmenting fungal killing than vesicles from encapsulated strains. Taken together, our findings suggest that fungal secretory vesicles have the potential to influence the interaction of C. neoformans with host cells.     

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

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

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Evaluation of Flagella and Flagellin of Pseudomonas aeruginosa as Vaccines

Pseudomonas aeruginosa is a serious pathogen in hospitalized, immunocompromised, and cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum made of polymerized flagellin proteins differentiated into two major serotypes: a and b. Antibodies to flagella delay onset of infection in CF patients, but whether immunity to polymeric flagella and that to monomeric flagellin are comparable has not been addressed, nor has the question of whether such antibodies might negatively impact Toll-like receptor 5 (TLR5) activation, an important component of innate immunity to P. aeruginosa. We compared immunization with flagella and that with flagellin for in vitro effects on motility, opsonic killing, and protective efficacy using a mouse pneumonia model. Antibodies to flagella were superior to antibodies to flagellin at inhibiting motility, promoting opsonic killing, and mediating protection against P. aeruginosa pneumonia in mice. Protection against the flagellar type strains PAK and PA01 was maximal, but it was only marginal against motile clinical isolates from flagellum-immunized CF patients who nonetheless became colonized with P. aeruginosa. Purified flagellin was a more potent activator of TLR5 than were flagella and also elicited higher TLR5-neutralizing antibodies than did immunization with flagella. Antibody to type a but not type b flagella or flagellin inhibited TLR5 activation by whole bacterial cells. Overall, intact flagella appear to be superior for generating immunity to P. aeruginosa, and flagellin monomers might induce antibodies capable of neutralizing innate immunity due to TLR5 activation, but solid immunity to P. aeruginosa based on flagellar antigens may require additional components beyond type a and type b proteins from prototype strains.Pseudomonas aeruginosa is an opportunistic pathogen responsible for a large proportion of ventilator-associated, hospital acquired pneumonia and is also a major cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum that has the added structural feature of being glycosylated (39). Flagellin is the primary protein component of the flagellar filament, and it can be classified into two serotypes, types a and b. Flagella carry out many functions, such as motility and attachment of bacteria to host cells, and can also elicit the activation of the host inflammatory response via Toll-like receptor 5 (TLR5) (6, 15, 29, 31). Importantly, promising results in terms of prevention of the acquisition of P. aeruginosa infection in CF patients immunized with a bivalent type a and b flagellum vaccine have been published (12).Several animal studies have not only demonstrated the importance of flagella as a virulence factor in P. aeruginosa but also validated them, or their flagellin component, as target antigens for vaccination. In the burned-mouse model of infection, chemically mutagenized or genetically produced flagellum-negative strains were less virulent than flagellum-positive strains (5, 26). It has also been shown with this model that motility is necessary for dissemination from the site of infection, since an intact flagellum structure is essential for death due to sepsis (5). In a neonatal model of acute P. aeruginosa pulmonary infection, flagella were essential for full virulence (14), although this was not found to be the case for adult mice with pulmonary P. aeruginosa infection (6). In regard to protection mediated by flagella or flagellin, immunization with flagella provided protection against infection and decreased the spread to major organs in the burned-mouse model (19). In a rat model of P. aeruginosa-induced pneumonia, administration of human monoclonal antibodies (MAbs) to flagella provided protection against infection and decreased lung injury (24), and another set of human MAbs provided protection in a murine model of pneumonia during neutropenia (28). A DNA vaccine encoding recombinant type a or type b P. aeruginosa flagellin also induced protective immunity against lethal P. aeruginosa lung infection (33), although this study curiously found better heterologous protection than homologous protection when DNA encoding wild-type flagellin was incorporated into the vaccine. A fusion protein of outer membrane protein F (OprF) residues 311 to 341, mature OprI residues 21 to 83, and flagellins a and b (termed OprF311-341-OprI-flagellins) generated significant immune responses in mice and promoted enhanced clearance of strain PA01 in a pulmonary challenge model (42). None of these studies directly compared the vaccine potential of flagellin with that of flagella.In addition to being highly immunogenic, the flagellin component of flagella serves as a pathogen-associated molecular pattern (PAMP), activating TLR5 and inducing innate immunity in the lung, stimulating a protective inflammatory response that contributes to the eradication of the pathogen (15, 32, 33, 35). Instillation of recombinant flagellin into the lungs of mice elicits a significant induction of innate immunity (20), and application of flagellin to the cornea of mice or intraperitoneal (i.p.) injection prior to corneal injury and local P. aeruginosa infection protects against pathological destruction of this tissue (22, 23). Finally, overexpression of flagellin monomers enhances virulence of P. aeruginosa (6).Of great interest is that the TLR5-binding domain of flagellin is not exposed in the intact flagella (36), and thus, flagellin monomers must be released or extracted from the intact flagella to promote TLR5 activation. Therefore, the comparative TLR5 agonist activity of flagellin, flagella, and even intact P. aeruginosa bacteria has not been evaluated, nor is it clear if the TLR5 activation component of flagellin would be immunogenic when immunizing with the intact polymeric flagella.Since P. aeruginosa serotype a and b flagella are conserved, contribute to virulence, stimulate innate immunity, and have induced protective efficacy in both animal (19, 24) and human (12) vaccine studies, it is clear that the flagellum or the flagellin monomer may be a useful target as a vaccine component, particularly as a carrier protein to link to protective carbohydrate antigens such as lipopolysaccharide (LPS) O-side chains or the alginate capsule (11, 30, 37). To our knowledge, no comparative analysis of the vaccine efficacy of flagellin versus that of flagella has been described for P. aeruginosa or other pathogens. Thus, it is not clear if it is flagellum or flagellin that is the best vaccine candidate, if either or both could be effectively utilized as a component of a conjugate vaccine, and if use of these vaccines could induce a state of enhanced susceptibility to infection by blocking flagellin-TLR5 interactions that promote effective innate immunity, as was found with antibodies induced by a DNA vaccine encoding P. aeruginosa flagellin (33). The purpose of this study was to compare whether immunity to P. aeruginosa flagella and that to flagellin are comparable or distinct and to evaluate if antibodies neutralizing TLR5 activation are induced and whether this impacted TLR5 activation by flagellin, flagella, or intact P. aeruginosa cells.     

Meningococcal Porin PorB Prevents Cellular Apoptosis in a Toll-Like Receptor 2- and NF-κB-Independent Manner

Meningococcal porin PorB is an inhibitor of apoptosis induced via the intrinsic pathway in various cell types. This effect is attributed to prevention of mitochondrial depolarization and of subsequent release of proapoptotic mitochondrial factors. To determine whether apoptosis is globally inhibited by PorB, we compared the intrinsic and extrinsic pathways in HeLa cells. Interestingly, PorB does not prevent extrinsic apoptosis induced by tumor necrosis factor alpha plus cycloheximide, suggesting a unique mitochondrial pathway specificity. Several intracellular factors regulated by NF-κB, including members of the Bcl-2 family and of the inhibitor of apoptosis (IAP) family, play major roles in controlling apoptosis, and some of them are thought to contribute to the antiapoptotic effect of the gonococcal porin, PIB. However, most of the members of the Bcl-2 family and the IAP family are not induced by meningococcal PorB in HeLa cells, with the exception of Bfl-1/A1. Interestingly, PorB does not induce NF-κB activation in HeLa cells, likely due to a lack of Toll-like receptor 2 (TLR2) expression in these cells. Bfl-1/A1 expression is also regulated by CBF1, a nuclear component of the Notch signaling pathway, independent of NF-κB activation. Since HeLa cells are protected by PorB from intrinsic apoptosis events, regardless of TLR2 and NF-κB expression, the possibility of a contribution of alternative signaling pathways to this effect cannot be excluded. In this paper, we describe an initial dissection of the cascade of cellular events involved in the antiapoptotic effect of PorB in the absence of TLR2.Apoptosis, or programmed cell death, is characterized by morphological events, including membrane blebbing and nuclear and chromatin condensation, and by intracellular events, such as activation of cytosolic proteins and DNA degradation (26). A variety of different intracellular stress signals can trigger apoptosis, including bacterial infections, excessive calcium, chemical substances, DNA-damaging agents (intrinsic or mitochondrial pathway), and cell surface death receptor activation (extrinsic pathway). Both pathways are divided into three basic phases: (i) initiation, (ii) commitment, and (iii) execution, ending with cell death (71).Several intracellular protein families, such as the Bcl-2 family (40), caspases (8), and the inhibitors of apoptosis (IAPs) (16), play important roles in controlling apoptosis. Bcl-2 proteins have a dual role; they trigger apoptosis (Bax, Bak, and Bid [1]) or block it (Bcl-2, Bcl-xL, Bfl-1, and Mcl-1 [39]). Proapoptotic Bcl-2 proteins can induce release of mitochondrial factors, including cytochrome c (36), apoptosis-inducing factor (AIF) (70), and Smac/DIABLO (18), in both a mitochondrial membrane potential-dependent manner and a mitochondrial membrane potential-independent manner (2, 25, 74). These events lead to activation of caspase 9 and 6 (intrinsic pathway) and subsequent DNA degradation. Alternatively, proapoptotic Bcl-2 proteins can also directly activate caspase 8 (extrinsic pathway) (1, 68), but the two pathways converge at a downstream event, caspase 3 activation (24). Antiapoptotic Bcl-2 proteins act mostly by modulating mitochondrial functions directly by interacting with mitochondrial components of the permeability transition pore or indirectly by neutralizing proapoptotic Bcl-2 proteins (7, 69, 72). IAPs are a family of proteins that directly inhibit caspase activation (16, 32) and, similar to Bcl-2 proteins, are also regulated by NF-κB (9).Modulation of apoptosis by several intracellular and extracellular bacteria, mostly to avoid normal host defense responses, has been described previously. Many bacteria induce and/or prevent apoptosis, depending on the host cell type, growth conditions, or bacterial life cycle. Some examples of bacteria that inhibit apoptosis are Chlamydia (20, 22, 79), Shigella flexneri (11), Brucella (28), Porphyromonas gingivalis (56, 58), Neisseria meningitidis, and Neisseria gonorrhoeae (4, 23, 31, 42, 50, 55, 62, 65, 73). Our group and other workers have reported that live N. meningitidis and purified meningococcal porin inhibit apoptosis (15, 49, 50, 62, 75), potentially via multiple mechanisms. While meningococcal infection induces NF-κB-mediated upregulation of antiapoptotic genes, purified PorB and PorB from live bacteria directly interact with mitochondria and modulate their membrane potential, preventing release of cytochrome c.N. gonorrhoeae and purified gonococcal porin PIB induce NF-κB-mediated upregulation of antiapoptotic genes (4, 5, 23, 33, 55, 65), which could also contribute to prevention of apoptosis.A correlation between the antiapoptotic effect of PorB and activation of NF-κB has not been shown so far, although our group has demonstrated that PorB activates NF-κB in a Toll-like receptor 2 (TLR2)-dependent manner (43, 46, 48, 52). Interestingly, various human and murine cell types are protected from apoptosis by PorB (23. 49, 50, 51) regardless of TLR2 expression. To clarify the role of this receptor in the antiapoptotic effect of PorB, this work focused in particular on naturally TLR2-deficient HeLa cells (78) and aimed at dissecting the potential cascade of cellular events elicited by PorB leading to protection from apoptosis.     

Tracking the Emerging Human Pathogen Pseudallescheria boydii by Using Highly Specific Monoclonal Antibodies

Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 κ-light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.Pseudallescheria boydii is an infectious fungal pathogen of humans (7, 16, 40, 58, 59). It is the etiologic agent of white grain mycetoma in immunocompetent humans (7) and has emerged over recent years as the cause of fatal disseminated infections in individuals with neutropenia, AIDS, diabetes, renal failure, bone marrow or solid organ transplants, systemic lupus erythematous, and Crohn''s disease; in those undergoing corticosteroid treatment; and in leukemia and lymphoma patients (1, 2, 3, 18, 27, 31, 32, 34, 36, 37, 38, 47, 49, 52). The fungus is the most prevalent species after Aspergillus fumigatus in the lungs of cystic fibrosis patients (8), where it causes allergic bronchopulmonary disease (5) and chronic lung lesions simulating aspergillosis (24). Near-drowning incidents and recent natural disasters, such as the Indonesian tsunami in 2004, have shown P. boydii and the related species Scedosporium apiospermum and Scedosporium aurantiacum to be the causes of fatal central nervous system infections and pneumonia in immunocompetent victims who have aspirated polluted water (4, 11, 12, 21, 22, 25, 30, 33, 57). Its significance as a potential pathogen of disaster evacuees has led to its recent inclusion in the Centers for Disease Control and Prevention list of infectious etiologies in persons with altered mental statuses, central nervous system syndromes, or respiratory illness.P. boydii is thought to be an underdiagnosed fungus (60), and misidentification is one of the reasons that the mortality rate due to invasive pseudallescheriasis is high. Detection of invasive P. boydii infections, based on cytopathology and histopathology, is problematic since it can occur in tissue and bronchoalveolar and bronchial washing specimens with other hyaline septated fungi, such as Aspergillus and Fusarium spp. (7, 23, 53, 60), which exhibit similar morphological characteristics upon microscopic examination (2, 23, 24, 28, 37, 44, 53, 60). Early diagnosis of infection by P. boydii and differentiation from other agents of hyalohyphomycosis is imperative, since it is refractory to antifungal compounds, such as amphotericin B, that are commonly administered for the control of fungal infections (10, 39, 58).The immunological diagnosis of Pseudallescheria infections has focused on the detection of antigens by counterimmunoelectrophoresis, and by immunohistological techniques using polyclonal fluorescent antibodies, but cross-reactions with antigens from other fungi, such as Aspergillus species, occurs (7, 19, 23). Pinto and coworkers (41, 42) isolated a peptidorhamnomannan from hyphae of P. boydii and proposed the antigen as a diagnostic marker for the pathogen. Cross-reactivity with Sporothrix schenckii and with Aspergillus have, however, been noted (23, 41). Furthermore, it is uncertain whether a similar antigen is present in the related pathogenic species S. prolificans, an important consideration in patient groups susceptible to mixed Scedosporium infections (6, 18).Hybridoma technology allows the production of highly specific MAbs that are able to differentiate between closely related species of fungi (54, 55, 56). The purpose of this paper is to report the development of MAbs specific to P. boydii and certain closely related species and their use to accurately discriminate among P. boydii, A. fumigatus, and other human pathogenic fungi by using immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs).Currently, the natural environmental habitat of P. boydii is unknown, but nutrient-rich, brackish waters, such as estuaries, have been suggested (9, 17). In combination with a semiselective isolation procedure, I show how the DAS-ELISA can be used to rapidly and accurately track the pathogen in naturally infested estuarine muds, and in doing so illustrate the potential of the DAS-ELISA as a diagnostic platform for detection of P. boydii and related species within the Pseudallescheria complex.     

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.     

Protection of Sheep against Rift Valley Fever Virus and Sheep Poxvirus with a Recombinant Capripoxvirus Vaccine

Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.Rift Valley fever (RFV) virus (RVFV) is a mosquito-borne member of the genus Phlebovirus, family Bunyaviridae. It is widely distributed in Africa, causing endemic and epidemic disease in both humans and livestock, including sheep, cattle, and goats. RVF was first described in Kenya and was shown to be caused by a filterable virus transmissible via blood (9). Acute RVF in lambs is characterized by fever and death within 24 to 48 h of being detected (43). Signs in adult sheep include fever, mucopurulent nasal discharge, hemorrhagic diarrhea, and abortion in pregnant ewes (43). RVFV can be transmitted from infected sheep to humans, particularly when humans are exposed to aborted sheep fetuses and blood.Attenuated live RVFV vaccines are available for use in livestock. A mutagen-attenuated RVFV vaccine induces protective immune responses in lambs and appears to be safe (25); however, other studies documented teratogenic effects on lambs from vaccinated pregnant ewes similar to those caused by the attenuated RVFV strain Smithburn (18). An inactivated RVFV vaccine induces neutralizing antibody responses in humans (33), and its use in sheep would not induce teratogenic effects or abortions. However, the inactivated vaccine requires 3 doses (33) and is expensive to produce. Efforts to make RVFV vaccines without these disadvantages include an attenuated RVFV developed by reverse genetics and lacking the NSs and NSm genes (4) and other new-generation RVFV vaccines (reviewed in reference 19) that protect mice against virus challenge (7, 16, 24, 27).The middle (M) RNA segment of the RVFV genome encodes the viral glycoproteins Gn and Gc (8, 20), and recombinant vaccinia virus expressing these glycoproteins induces neutralizing antibody and protective immunity to RVFV in mice (7). Vaccinia virus is safe for animals, but there is some risk to humans, as it was reported previously to spread from human vaccinees to contacts (28, 55) and to cause serious clinical disease in human immunodeficiency virus-infected patients (36). Although modified vaccinia virus Ankara is a safer alternative for humans (6, 57), there are animal poxviruses with naturally restricted host ranges for vaccine vectors in animals (1, 13, 30, 31, 40, 46, 47, 52, 53).For ruminants, the genus Capripoxvirus (CPV) of the family Poxviridae has been an effective recombinant vector to induce protective immunity against several other viruses (3, 17, 29, 32, 40, 41, 51). This genus has three closely related species causing sheep pox, goat pox, and lumpy skin disease (LSD) of cattle. A recombinant LSD vaccine expressing the Gn and Gc glycoproteins of RVFV induced protection against RVFV challenge in mice (52, 53) and sheep (52). The three species of CPV have 96 to 97% nucleotide identity (49) and are restricted to ruminants, with no evidence of human infections (10, 11). Furthermore, attenuated CPV vaccines are in use in Africa and the Middle East to control ruminant poxvirus disease (11, 21). The use of a CPV vector to deliver virus vaccines to ruminants also induces immunity to the CPV vector, thus increasing the valence of the vaccine (3, 17, 39, 40). We report here the construction of a recombinant CPV that expresses the RVFV Gn and Gc glycoproteins and induces protective immunity against RVFV and sheep poxvirus (SPV) challenge in sheep.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.