Role of B Cells in Host Defense against Primary Coxiella burnetii Infection

Despite Coxiella burnetii being an obligate intracellular bacterial pathogen, our recent study demonstrated that B cells play a critical role in vaccine-induced immunity to C. burnetii infection by producing protective antibodies. However, the role of B cells in host defense against primary C. burnetii infection remains unclear. In this study, we investigated whether B cells play an important role in host defense against primary C. burnetii infection. The results showed that peritoneal B cells were able to phagocytose virulent C. burnetii bacteria and form Coxiella-containing vacuoles (CCVs) and that C. burnetii can infect and replicate in peritoneal B1a subset B cells in vitro, demonstrating a potential role for peritoneal B cells in host defense against C. burnetii infection in vivo. In addition, the results showing that B1a cells secreted a high level of interleukin-10 (IL-10) in response to C. burnetii infection in vitro suggest that B1a cells may play an important role in inhibiting the C. burnetii infection-induced inflammatory response. The observation that adoptive transfer of peritoneal B cells did not significantly affect the severity of C. burnetii infection-induced diseases in both severe combined immunity-deficient (SCID) and μMT mice indicates that peritoneal B cells alone may not be able to control C. burnetii infection. In contrast, our finding that C. burnetii infection induced more-severe splenomegaly and a higher bacterial burden in the spleens of B1a cell-deficient Bruton''s tyrosine kinase x-linked immunity-deficient (BTK^xid) mice than in their wild-type counterparts further suggests that B1a cells play an important role in host defense against primary C. burnetii infection.     

Apoptosis in Macrophages and Alveolar Epithelial Cells during Early Stages of Infection by Legionella pneumophila and Its Role in Cytopathogenicity

The hallmark of Legionnaires’ disease is intracellular replication of Legionella pneumophila within cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection by L. pneumophila are not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells by L. pneumophila during early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis in L. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellular L. pneumophila is capable of inducing apoptosis. Furthermore, induction of apoptosis by L. pneumophila correlates with cytopathogenicity. We conclude that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection.     

Role of Dectin-2 for Host Defense against Systemic Infection with Candida glabrata

Although Candida glabrata is an important pathogenic Candida species, relatively little is known about its innate immune recognition. Here, we explore the potential role of Dectin-2 for host defense against C. glabrata. Dectin-2-deficient (Dectin-2^−/−) mice were found to be more susceptible to C. glabrata infections, showing a defective fungal clearance in kidneys but not in the liver. The increased susceptibility to infection was accompanied by lower production of T helper 1 (Th1) and Th17-derived cytokines by splenocytes of Dectin-2^−/− mice, while macrophage-derived cytokines were less affected. These defects were associated with a moderate yet significant decrease in phagocytosis of the fungus by the Dectin-2^−/− macrophages and neutrophils. Neutrophils of Dectin-2^−/− mice also displayed lower production of reactive oxygen species (ROS) upon challenge with opsonized C. glabrata or C. albicans. This study suggests that Dectin-2 is important in host defense against C. glabrata and provides new insights into the host defense mechanisms against this important fungal pathogen.     

Dendritic Cell-Based Immunization Ameliorates Pulmonary Infection with Highly Virulent Cryptococcus gattii

Cryptococcosis due to a highly virulent fungus, Cryptococcus gattii, emerged as an infectious disease on Vancouver Island in Canada and surrounding areas in 1999, causing deaths among immunocompetent individuals. Previous studies indicated that C. gattii strain R265 isolated from the Canadian outbreak had immune avoidance or immune suppression capabilities. However, protective immunity against C. gattii has not been identified. In this study, we used a gain-of-function approach to investigate the protective immunity against C. gattii infection using a dendritic cell (DC)-based vaccine. Bone marrow-derived dendritic cells (BMDCs) efficiently engulfed acapsular C. gattii (Δcap60 strain), which resulted in their expression of costimulatory molecules and inflammatory cytokines. This was not observed for BMDCs that were cultured with encapsulated strains. When Δcap60 strain-pulsed BMDCs were transferred to mice prior to intratracheal R265 infection, significant amelioration of pathology, fungal burden, and the survival rate resulted compared with those in controls. Multinucleated giant cells (MGCs) that engulfed fungal cells were significantly increased in the lungs of immunized mice. Interleukin 17A (IL-17A)-, gamma interferon (IFN-γ)-, and tumor necrosis factor alpha (TNF-α)-producing lymphocytes were significantly increased in the spleens and lungs of immunized mice. The protective effect of this DC vaccine was significantly reduced in IFN-γ knockout mice. These results demonstrated that an increase in cytokine-producing lymphocytes and the development of MGCs that engulfed fungal cells were associated with the protection against pulmonary infection with highly virulent C. gattii and suggested that IFN-γ may have been an important mediator for this vaccine-induced protection.     

A Role for Interleukin-6 in Host Defense against Murine Chlamydia trachomatis Infection

Interleukin-6-deficient (IL-6^−/−) knockout mice had significantly increased Chlamydia trachomatis levels in lung tissue and increased mortality compared to B6129F₂/J controls early after intranasal infection. Gamma interferon production and chlamydia-specific antibody levels were consistent with a decreased but reversible Th1-like response in IL-6^−/− mice. IL-6 is needed for an optimal early host response to this infection.     

Role of NK Cells in Early Host Response to Chlamydial Genital Infection

The cell-mediated immune response has been documented to be the major protective immune mechanism in mice infected genitally with the agent of mouse pneumonitis (MoPn), a biovar of Chlamydia trachomatis. Moreover, there is strong evidence to indicate that gamma interferon (IFN-γ) is a major effector mechanism of the cell-mediated immune response. Previous studies from this laboratory have also reported that the dominant cell population in the genital tract is the CD4 Th1 population. When experiments were performed by the enzyme-linked immunospot assay, high numbers of cells producing IFN-γ were found in the genital tract, concomitant with resolution of the infection; however, in addition, an increase in IFN-γ-producing cells which were CD4⁻ was seen early in the infection. Since natural killer (NK) cells produce IFN-γ and have been found to participate in the early responses in other infections, we hypothesized that NK cells are responsible for early IFN-γ production in the murine chlamydial model. NK cells were quantified by the standard YAC-1 cytotoxicity assay and were found to appear in the genital tract as early as 12 h after intravaginal infection with MoPn. The cells were confirmed to be NK cells by abrogation of YAC-1 cell cytotoxicity by treatment in vitro and in vivo with anti-asialo-GM1. The early IFN-γ response could also be depleted by treatment with anti-asialo-GM1, indicating that NK cells were responsible for the production of this cytokine. Of interest was our observation that depletion of NK cells also exacerbated the course of infection in the mice and elicited a Th2 response, as indicated by a marked increase in immunoglobulin G1 antibody. Thus, these data demonstrate that NK cells are not only responsible for the production of IFN-γ early in the course of chlamydial genital tract infection but are also, via IFN-γ, a significant factor in the development of the Th1 CD4 response and in the control of the infection.     

Fbp1-Mediated Ubiquitin-Proteasome Pathway Controls Cryptococcus neoformans Virulence by Regulating Fungal Intracellular Growth in Macrophages

Cryptococcus neoformans is a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential for Cryptococcus virulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that the fbp1Δ mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. The fbp1Δ mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of the fbp1Δ mutant is due to its inability to leave the lung. The fbp1Δ mutant showed a defect in intracellular proliferation after phagocytosis in a Cryptococcus-macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulating Cryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis.     

Role of Complement in Protection against Cryptococcus gattii Infection

Previous studies have shown that the alternative pathway of complement activation plays an important role in protection against infection with Cryptococcus neoformans. Cryptococcus gattii does not activate the alternative pathway as well as C. neoformans in vitro. The role of complement in C. gattii infection in vivo has not been reported. In this study, we used mice deficient in complement components to investigate the role of complement in protection against a C. gattii isolate from an ongoing outbreak in northwestern North America. While factor B-deficient mice showed an enhanced rate of death, complement component C3-deficient mice died even more rapidly, indicating that the alternative pathway was not the only complement pathway contributing to protection against disease. Both C3- and factor B-deficient mice had increased fungal burdens in comparison to wild-type mice. Histopathology revealed an overwhelming fungal burden in the lungs of these complement-deficient mice, which undoubtedly prevented efficient gas exchange, causing death. Following the fate of radiolabeled organisms showed that both factor B- and C3-deficient mice were less effective than wild-type mice in clearing organisms. However, opsonization of C. gattii with complement components was not sufficient to prolong life in mice deficient in complement. Killing of C. gattii by macrophages in vitro was decreased in the presence of serum from factor B- and C3-deficient versus wild-type mice. In conclusion, we have demonstrated that complement activation is crucial for survival in C. gattii infection. Additionally, we have shown that the alternative pathway of complement activation is not the only complement pathway contributing to protection.The complement system consists of a cascade of serum proteins that are involved in opsonization, membrane lysis, and chemotaxis. There are three pathways through which complement can be activated: classical, alternative, and lectin. Complement components C3 to C9 participate in all three complement cascade pathways. C1q is used only in the classical pathway, and factor B is used only in the alternative pathway. While C4 is used in both the classical and lectin pathways, recently it was reported that the lectin pathway can function in the absence of C2 and/or C4 if the alternative pathway is intact (23). Mannose binding lectin (MBL) (24), which is used only in the lectin pathway, exists in two forms in rodents (MBL-A and MBL-C) while only one form is found in humans (8). MBL-A and MBL-C have different levels in serum and differ in their affinity for d-glucose and α-methyl-d-glucose but have redundant function (24).Cryptococcus spp. are fungal pathogens that possess a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM). The capsule is antiphagocytic and anti-inflammatory but is known to activate the alternative complement pathway (11). Previously, Cryptococcus spp. were identified serologically and were all considered as one species, Cryptococcus neoformans. More recently, two species have been designated, C. neoformans (serotypes A and D) and Cryptococcus gattii (serotypes B and C). A key difference between the two species is that C. neoformans tends to infect immunocompromised individuals while C. gattii generally infects apparently immunocompetent people (15). Cryptococcus spp. most commonly cause pulmonary and central nervous system infections in humans (15, 25) and mouse models (3).In 1999, an outbreak of C. gattii began on Vancouver Island, British Columbia, infecting people, companion animals, and porpoises. Strain A1MR265 is the major clinical reference isolate from the Vancouver Island outbreak. Recently, a case of cryptococcosis caused by the strain predominant in Vancouver Island was identified in Puget Sound, WA (27). A total of eight human cases have been reported in Washington State in the past 2 years; four of these individuals had not traveled out of state (29). Nine cases have been reported in Oregon (L. Hoang, presented at the 108th General Meeting of the American Society of Microbiology, Boston, MA, 1 to 5 June 2008). The potential spreading of a strain of Cryptococcus capable of infecting immunocompetent people is cause for concern, and C. gattii infection is now a reportable disease in Washington State (29).The Kozel laboratory and others have shown that Cryptococcus spp. strongly activate the alternative pathway of the complement cascade (5, 14) while the polysaccharide capsule blocks the activation of the classical pathway that occurs at the cell wall in nonencapsulated strains (13). The capsule serves as a site for activation and deposition of C3 fragments, mainly iC3b, which promote phagocytosis of the yeast (12). C. gattii does not appear to activate the alternative pathway as potently as C. neoformans (28, 31). One study found that C. gattii binds fewer C3 molecules than C. neoformans (28). A later report indicated that while the maximum amount of bound C3 did not differ significantly between species, there was more rapid accumulation of C3 on C. neoformans before a steady state was achieved (31). In the absence of C5 in mouse strains B10.D2/oSn, DBA/2, and A/J, C. neoformans infection proceeds to a fatal pneumonia with higher fungal burdens in the blood, brain, lungs, and liver than in complement-sufficient animals (6, 21, 22). Together, these studies indicate a significant role for the alternative pathway of complement in protection against C. neoformans. While C. gattii has been reported to not activate the alternative pathway as vigorously as C. neoformans in vitro, the role of complement in in vivo C. gattii infection has not been determined.In this study, we investigated the role of complement pathways in protecting against infection with C. gattii. Mice deficient in complement components C1q, C4, C3, and factor B have been generated on the C57BL/6J background (2, 16, 30). Using these mice and mice treated with cobra venom factor (CVF), which depletes C3 and prevents cascade progression from C3 to C9, we have now shown that complement activation plays an essential role in delaying disease progression in mice infected with C. gattii.     

Role of NADPH Phagocyte Oxidase in Host Defense against Acute Respiratory Acinetobacter baumannii Infection in Mice

Acinetobacter baumannii is an emerging bacterial pathogen that rapidly develops multiple-drug resistance and is responsible for many nosocomial pulmonary infections. This study investigated the role of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (NOS2) in the host defense against respiratory infection with A. baumannii in mouse models of intranasal A. baumannii infection. gp91^phox−/− mice showed higher susceptibility to A. baumannii infection than wild-type (WT) C57BL/6 mice, with significantly greater bacterial counts in their lungs (1,000-fold) (P < 0.005) and spleens (10-fold) (P < 0.05). Moreover, all of the gp91^phox−/− mice succumbed to infection within 48 h. In contrast, only a moderate increase in bacterial burdens was detected in the lungs of NOS2^−/− mice, and all NOS2^−/− mice survived infection. Compared to WT mice, the pulmonary influx of inflammatory cells and serum and local inflammatory cytokine/chemokine responses were not obviously impaired at 4 h and were significantly higher at 24 h (P < 0.05) in gp91^phox−/− mice, but NADPH-deficient neutrophils were unable to control bacterial replication and extrapulmonary dissemination. Thus, NADPH phagocyte oxidase appears to play a crucial role in the neutrophil-mediated host defense against A. baumannii.Acinetobacter baumannii infection has recently emerged as a major cause of both community-associated and nosocomial infections worldwide (6, 7). The overall 30-day mortality of Acinetobacter infection can be as high as 49%, with the respiratory tract being an important portal of entry (12). Moreover, A. baumannii infections are becoming increasingly difficult to treat because of its rapid development of resistance to multiple antibiotics (4). Despite its clinical importance, little is known regarding the host defense mechanisms against respiratory A. baumannii infection.We and others have recently shown that C57BL/6 mice can effectively control and eliminate a sublethal A. baumannii infection within 72 h following intranasal inoculation, which requires an influx of neutrophils into the lung but does not require gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) (8, 10, 19, 22). The depletion of neutrophils with monoclonal antibody (22) or cyclophosphamide (8) significantly enhanced bacterial multiplication and converted an otherwise self-limiting infection into a fatal infection, suggesting that neutrophils are crucial in the control of local bacterial multiplication and subsequent extrapulmonary dissemination. However, the mechanisms by which neutrophils inhibit or kill A. baumannii remain unknown.Neutrophils can contribute to host innate defense mechanisms through the production of reactive oxygen species (ROS) (11, 16, 17). Activated neutrophils produce ROS primarily through NADPH phagocyte oxidase catalyzation, which leads to the production of superoxide, which is the precursor to more toxic ROS including H₂O₂ and OH (14, 23). Also, stimulated neutrophils and macrophages release myeloperoxidase from cytoplasmic granules to interact with H₂O₂ to form hypohalous acids, which are very potent oxidants that can disrupt bacterial membrane integrity, normal metabolism, and replication (15). Neutrophils can also contribute to host innate defense mechanisms through the production of reactive nitrogen species (RNS) (11, 16, 17). Inducible nitric oxide synthase, also called NOS2, is an oxidant synthase found in the immune system that catalyzes the formation of NO and the subsequent auto-oxidation of a variety of RNS such as NO₂ and N₂O₃, which have substantially more antimicrobial activity (14, 23). In addition, NOS2 and NADPH oxidase can act synergistically to form highly potent radicals, including ONOO⁻, which can damage lipids, proteins, and DNA (18).In this study, we examined the roles of NADPH phagocyte oxidase and NOS2 in limiting bacterial replication during respiratory A. baumannii infection by using gp91^phox−/− and NOS2^−/− mice. We found that NADPH-dependent antimicrobial action is essential for an effective host defense against acute respiratory A. baumannii infection.     

Isotype Switching Increases Efficacy of Antibody Protection against Cryptococcus neoformans Infection in Mice

The isotype and epitope specificities of antibodies both contribute to the efficacy of antibodies that mediate immunity to Cryptococcus neoformans, but the relationship between these properties is only partially understood. In this study, we analyzed the efficacy of protection of two sets of immunoglobulin G (IgG) isotype switch variants from two IgG3 monoclonal antibodies (MAbs) which are either not protective or disease enhancing, depending on the mouse model used. The two IgG3 MAbs 3E5 and 4H3 have different epitope specificities. Protection experiments were done with A/JCr mice infected intravenously with C. neoformans and administered with 3E5 IgG3 and its IgG1, IgG2a, and IgG2b switch variants. These experiments revealed that IgG1, IgG2b, and IgG2a were each more effective than IgG3. For 4H3 IgG3 and its IgG1 and IgG2b switch variants, the relative efficacy was IgG2b > IgG1 >> IgG3. The combination of 3E5 IgG3 and 4H3 IgG3 was more deleterious than either IgG3 alone. All IgG isotypes were opsonic for mouse bronchoalveolar cells, with the relative efficacy being IgG2b > IgG2a > IgG1 > IgG3. These results (i) confirm that a nonprotective IgG3 MAb can be converted to a protective MAb by isotype switching, (ii) indicate that the efficacy of protection of an IgG1 MAb can be increased by isotype switching to another subclass, (iii) show that protective and nonprotective IgG MAbs are opsonic, and (iv) provide additional evidence for the concept that the efficacy of the antibody response to C. neoformans is dependent on the type of MAb elicited.Cryptococcus neoformans is a fungus which is a frequent cause of life-threatening meningoencephalitis in patients with impaired immunity (22, 25). Cryptococcosis has been reported to occur in 6 to 8% of patients with AIDS (7). In immunocompromised individuals, C. neoformans infections are often incurable with conventional antifungal agents, and these patients frequently require lifelong therapy (45). The difficulties involved in the management of cryptococcosis in immunocompromised individuals have led to a reexamination of the potential of antibody-mediated immunity for prevention and therapy of cryptococcal infections. A polysaccharide-tetanus toxoid (TT) conjugate vaccine which is highly immunogenic and can elicit protective antibodies in mice has been made (3, 8, 9). In addition, several monoclonal antibodies (MAbs) have been shown to modify the course of infection in mice, and these may be useful in therapy of human infection (12, 14, 28, 42, 43).Cell-mediated immunity is generally acknowledged to provide important host defense against C. neoformans infection (4, 20, 26, 31, 42). In contrast, the role of antibody-mediated immunity in host resistance is less certain (2), but there is considerable evidence that administration of some MAbs can modify the course of infection in mice (8, 12, 14, 16, 28, 33). C. neoformans is unusual among fungal pathogens in that it has a polysaccharide capsule composed primarily of glucuronoxylomannan (GXM) (6), which is important for virulence (5). The capsular polysaccharide has been shown to produce a variety of deleterious effects including inhibition of phagocytosis (21), interference with antigen presentation (39), shedding of adhesion molecules (11), inhibition of leukocyte migration (10), and alterations in cytokine production by host effector cells (24, 40, 41). Antibodies to the C. neoformans capsular polysaccharide may contribute to host defense through multiple effects including enhanced opsonization (13, 18, 23, 30, 44), clearance of polysaccharide antigen (15), promotion of granuloma formation (14), and release of oxygen- and nitrogen-derived oxidants (27, 38).In previous studies, we demonstrated that immunoglobulin G3 (IgG3) MAbs are not protective in various mouse models of cryptococcal infection (32, 42). When one of these nonprotective IgG3 MAbs was switched to IgG1, the IgG1 significantly prolonged animal survival (32, 42). In the present study, we analyzed two families of IgG switch variants generated in vitro from two nonprotective IgG3 MAbs with different epitope specificities. We found that MAbs with different isotypes have different protective efficacies and that switching of nonprotective IgG3 MAbs to IgG1, IgG2b, and IgG2a significantly increased antibody protective efficacy. These studies demonstrate a complex relationship among efficacy of antibody protection, epitope specificity, and isotype.     

Cryptococcus neoformans Resides in an Acidic Phagolysosome of Human Macrophages

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2′,7′-difluorofluorescein (Oregon Green). These probes have pK_as of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 μM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.     

Macrophages and Dendritic Cells in Antigen-Induced Arthritis

Two non-lymphoid cell types play an important role in the pathogenesis of arthritis, i.e. the 'classical' macrophage and the antigen-presenting dendritic cell. In the present study, the roles of both cell types are studied in antigen-induced arthritis of the rat knee joint. Cryostat sections of whole, unfixed, undecalcified knee joints were used for immunohistochemical staining of non-lymphoid cells and lymphocyte subsets. For the demonstration of the different types of non-lymphoid cells, monoclonal antibodies against rat macrophages (ED1, ED2, and ED3) and against Ia antigen were used with an immunoperoxidase method. The results show in an overall view of the arthritic joint the different sites of action of the classical macrophages on the one hand and the Ia-positive dendritic cells on the other. Classical macrophages were mainly found in the superficial layers of the synovium bordering joint space and articular cartilage. Dendritic cells and T cells of the helper phenotype were mainly found in clusters surrounding small blood vessels within the synovium. These clusters express the immunological background of the antigen-induced arthritis and may well be responsible for the continuation of the arthritic process.     

Role of Complement in Host Defense against Pneumococcal Otitis Media

Strategies to limit complement deposition on Streptococcus pneumoniae are established as virulence features for invasive disease, but their role in respiratory tract infection requires further analysis. We evaluated complement C3 protein deposition on discordant S. pneumoniae isolates of the same serotype (6A) and their capacity to cause nasopharyngeal (NP) colonization and experimental otitis media (EOM) in an animal model. We compared C3 binding to five 6A isolates from asymptomatic NP carriers with five 6A strains that caused invasive disease, and we observed less C3 (∼10-fold less fluorescence) binding to invasive isolates. We selected two high-level C3-binding carriage and two low-level C3-binding invasive 6A isolates for further study. In the EOM model, 11/12 (92%) ears challenged with a low-level C3-binding 6A strain became infected. Only 2/8 (25%) ears challenged with the discordant high-level C3-binding 6A isolate developed disease (P = 0.005). Results with the second discordant 6A isolate pair were comparable. Cobra venom factor (CoVF) treatment, which depletes C3 and consumes complement, restored virulence of the high-level C3-binding strain; 8/8 (100%) ears in CoVF-treated animals developed EOM compared to only 25% of ears in naïve animals (P = 0.007). These studies demonstrate the critical role for complement evasion in pneumococcal EOM. Colonization with carriage isolates that bound high levels of C3 caused EOM in fewer animals compared to low-level C3-binding invasive strains. Thus, limiting C3 deposition on the surface of S. pneumoniae correlates with increased incidence of EOM following NP colonization and barotrauma in the animal model.The pathogenesis of Streptococcus pneumoniae infection involves initial colonization of the nasopharynx, followed by its spreading to the middle ear, sinus, or lower respiratory tract and, in some cases, invasion of the bloodstream. To successfully cause disease, the pneumococcus has evolved a number of mechanisms to avert complement-mediated opsonization and phagocytosis. Pneumococci possess a broad variety of specialized surface proteins, some of which are adapted to interact with host defenses during colonization or dissemination in humans. Being a gram-positive bacterium, it is resistant to the bactericidal activity of complement (24) because its rigid cell wall prevents lysis by the membrane attack complex. The capsular polysaccharide is critical for resistance to complement deposition (32) and may also mask cell wall-associated complement from being recognized by the complement receptors on phagocytes (6). Additionally, select surface proteins can degrade native C3 proteins, thereby preventing or diminishing binding of C3b and iC3b to the bacterial surface, which are necessary components for opsonization (3). Furthermore, an important role for complement is suggested by the association of increased risk for invasive infections in individuals (or animal models) with deficiencies of complement proteins such as C2 and C3 and of complement receptors such as CR3 (2, 16). Type-specific antibody formation is an important host defense mechanism against infections caused by S. pneumoniae. However, the efficacy of opsonization of pneumococci by either immunoglobulin M (IgM) or IgG is related to their ability to enhance complement deposition on the bacterial surface, thus making complement essential for recovery from pneumococcal disease (6, 9).Colonization of mucosal surfaces is often the first step in the development of disease. Studies of S. pneumoniae support recent acquisition as the critical event preceding the development of pneumococcal otitis media. S. pneumoniae has evolved specific characteristics that are critical in dictating initial success for establishing colonization within a competitive niche of the mucosal surface of the nasopharynx. Often the success of an organism in establishing carriage depends on its ability to resist innate clearance mechanisms generated in the setting of polymicrobial stimulation. Lysenko and colleagues have demonstrated that complement and polymorphonuclear leukocytes are necessary host defenses for the elimination of S. pneumoniae from the nasopharynx in the presence of nontypeable Haemophilus influenzae (NTHi) (29).Prior work from our laboratory demonstrated that complement was an important host defense mechanism against protection of the middle ear from infection with NTHi (15). We hypothesized that complement would also be relevant for protection against S. pneumoniae respiratory tract infection (RTI). We evaluated the role of complement by comparing the capacities of four isolates of S. pneumoniae, all belonging to serotype 6A but differing in their abilities to bind complement to their surface to cause otitis media following nasopharyngeal (NP) colonization. Our goal was to determine whether evasion of complement deposition was an important virulence feature in the pathogenesis of RTI, using a model for experimental otitis media (EOM). The model requires initial NP colonization followed by ascension through the Eustachian tube after barotrauma for establishing middle ear infection.     

Dectin-2 Deficiency Promotes Th2 Response and Mucin Production in the Lungs after Pulmonary Infection with Cryptococcus neoformans

Dectin-2 is a C-type lectin receptor that recognizes high mannose polysaccharides. Cryptococcus neoformans, a yeast-form fungal pathogen, is rich in polysaccharides in its cell wall and capsule. In the present study, we analyzed the role of Dectin-2 in the host defense against C. neoformans infection. In Dectin-2 gene-disrupted (knockout) (Dectin-2KO) mice, the clearance of this fungus and the inflammatory response, as shown by histological analysis and accumulation of leukocytes in infected lungs, were comparable to those in wild-type (WT) mice. The production of type 2 helper T (Th2) cytokines in lungs was higher in Dectin-2KO mice than in WT mice after infection, whereas there was no difference in the levels of production of Th1, Th17, and proinflammatory cytokines between these mice. Mucin production was significantly increased in Dectin-2KO mice, and this increase was reversed by administration of anti-interleukin 4 (IL-4) monoclonal antibody (MAb). The levels of expression of β1-defensin, cathelicidin, surfactant protein A (Sp-A), and Sp-D in infected lungs were comparable between these mice. In in vitro experiments, IL-12p40 and tumor necrosis factor alpha (TNF-α) production and expression of CD86 and major histocompatibility complex (MHC) class II by bone marrow-derived dendritic cells and alveolar macrophages were completely abrogated in Dectin-2KO mice. Finally, the disrupted lysates of C. neoformans, but not of whole yeast cells, activated Dectin-2-triggered signaling in an assay with nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Dectin-2 may oppose the Th2 response and IL-4-dependent mucin production in the lungs after infection with C. neoformans, and it may not be required for the production of Th1, Th17, and proinflammatory cytokines or for clearance of this fungal pathogen.     

Characteristics of Invasive Candidiasis in Gamma Interferon- and Interleukin-4-Deficient Mice: Role of Macrophages in Host Defense against Candida albicans

Murine models of invasive candidiasis were used to study the in vivo importance of gamma interferon (IFN-γ) and interleukin-4 (IL-4) in host defense against Candida albicans and to characterize the tissue inflammatory reactions, with special reference to macrophages (Mφ). Knockout (KO) IFN-γ-deficient (GKO) and IL-4-deficient (IL-4 KO) and C57BL/6 parental mouse strains were challenged intraperitoneally with 10⁸ C. albicans blastoconidia. Survival of GKO mice was significantly lower (16.7%) than that of C57BL/6 control (55.5%) and IL-4 KO (61.1%) animals, but was not correlated with the extent of organ colonization. Immunohistological analysis with a panel of myeloid and lymphoid markers revealed multiple renal abscesses, myocarditis, hepatitis, meningoencephalitis, and pneumonia in each strain, with a dominant presence of Mφ. In the absence of IFN-γ, C. albicans induced striking changes in the phenotype of alveolar Mφ and extensive perivascular lymphoid infiltrates in the lung. Impairment in nitric oxide production by peritoneal Mφ was shown only in GKO mice, and they produced Candida-specific immunoglobulin G (IgG), IgM, IgA, and IgG subclasses in lower titers. Our in vivo studies with KO mice elucidate a critical role for IFN-γ, but not IL-4, in host defense against C. albicans.Candida albicans is a common commensal organism in humans, and its importance as an opportunistic pathogen, particularly in immunocompromised patients, has continued to increase over the last two decades. According to the National Nosocomial Infections Surveillance System, the ratio of C. albicans isolates among nosocomial fungal infections increased from 52% to 63% in the 1980s (4). Phagocytic cell defects generally predispose to disseminated candidiasis; candidemia was calculated to result in 38% excess mortality and extend hospitalization by approximately 30 days (40). Besides the efforts to develop more effective and safer antifungal agents, a new therapeutic approach to augment the antifungal capacity of the host’s immune system should be investigated.The mechanisms of host defense and pathogenesis of candidiasis are not completely understood. Optimal phagocytosis of C. albicans requires opsonization; however, unopsonized yeast can be internalized by macrophages (Mφ) through the mannose receptor (21). Efficient killing of C. albicans by mononuclear phagocytes requires respiratory burst-associated toxic compounds (22), and recent data suggest that nitric oxide (NO) may also be involved in anticandidal functions of Mφ (5). Experimental evidence suggests that mononuclear phagocytes could play an important role in eradication of this pathogen, and their anticandidal activity can be augmented in vitro with granulocyte-Mφ and Mφ colony-stimulating factors and cytokines (no significant change could be measured in the level of specific immunoglobulin A [IgA] in serum or among the levels of interleukin-3 [IL-3] and gamma interferon [IFN-γ]) in both human and murine systems (23, 25, 28, 39).The in vivo benefit of cytokine treatment in disseminated candidiasis has not been established, and data from different murine models are controversial. Administration of IFN-γ has been reported to be associated with improved survival of mice after lethal challenge with C. albicans, which correlated with the anticandidal activity of peritoneal Mφ (28); another study showed a reduction in tissue fungal burden in IFN-γ-treated mice (19). However, in a different murine model, in vivo administration of IFN-γ resulted in increased susceptibility and organ colonization of four infected inbred strains (13). In vivo administration of IL-12, which has been reported to prime naive T cells for high IFN-γ expression and skew cytokine production toward a Th1-type response (38), did not modify the course of systemic candidiasis (32). In contrast, Th2-type cytokines IL-4 and IL-10 have been reported to exacerbate infection, and neutralization of IL-4 by specific antibody or soluble IL-4 receptor resulted in an enhanced production of Th1 cytokines, associated with increased resistance to systemic murine candidiasis (26, 30, 37). The controversial results of in vivo cytokine treatment may be the consequence of genetic differences among the infected strains and also the variation in protocols; the kinetics of cytokine production are influenced by several host and pathogen factors, and the effect of exogenous cytokine might depend on the condition of the infected host and stage of infection.Cytokine and receptor gene disruption strategies make it possible to examine the role of cytokines in host response to different pathogens directly. Recent studies showed an increased susceptibility of IFN-γ–receptor knockout (KO) mice to Mycobacterium bovis or Mycobacterium tuberculosis, but not to Schistosoma mansoni (1, 7, 8). Another study reported that disruption of the IFN-γ receptor gene was associated with higher susceptibility to Leishmania major and that IL-4 deficiency resulted in increased resistance, but only in certain inbred strains (17).Our study was undertaken to investigate the in vivo role of IFN-γ and IL-4 in disseminated C. albicans infection and characterize the tissue inflammatory cells by immunohistochemistry and by functional assays ex vivo. We demonstrate that IFN-γ, but not IL-4, is essential for survival in invasive candidiasis and show the dominant participation of Mφ in the inflammatory lesions of different tissues in KO as well as wild-type mice. In the absence of IFN-γ, a striking local immune regulatory alteration was observed in the lungs.     

Defect of CARD9 Leads to Impaired Accumulation of Gamma Interferon-Producing Memory Phenotype T Cells in Lungs and Increased Susceptibility to Pulmonary Infection with Cryptococcus neoformans

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule signal that is critical for NF-κB activation and is triggered through C-type lectin receptors (CLRs), which are pattern recognition receptors that recognize carbohydrate structures. Previous studies have reported that Cryptococcus neoformans, a fungal pathogen that causes meningoencephalitis in AIDS patients, is recognized through some CLRs, such as mannose receptors or DC-SIGN. However, the role of CARD9 in the host defense against cryptococcal infection remains to be elucidated. In the present study, we analyzed the role of CARD9 in the host defense against pulmonary infection with C. neoformans. CARD9 gene-disrupted (knockout [KO]) mice were highly susceptible to this infection, as shown by the reduced fungal clearance in the infected lungs of CARD9 KO mice, compared to that in wild-type (WT) mice. Gamma interferon (IFN-γ) production was strongly reduced in CARD9 KO mice during the innate-immunity phase of infection. Reduced IFN-γ synthesis was due to impaired accumulation of NK and memory phenotype T cells, which are major sources of IFN-γ innate-immunity-phase production; a reduction in the accumulation of these cells was correlated with reduced CCL4, CCL5, CXCL9, and CXCL10 synthesis. However, differentiation of Th17 cells, but not of Th1 cells, was impaired at the adaptive-immunity phase in CARD9 KO mice compared to WT mice, although there was no significant difference in the infection susceptibility between interleukin 17A (IL-17A) KO and WT mice. These results suggest that CARD9 KO mice are susceptible to C. neoformans infection probably due to the reduced accumulation of IFN-γ-expressing NK and memory phenotype T cells at the early stage of infection.