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Genes and Immunity (2004) 5, 576–587 & 2004 Nature Publishing Group All rights reserved 1466-4879/04 $30.00 www.nature.com/gene FULL PAPER Genetic analysis of innate immunity in resistance to Candida albicans A Tuite1, A Mullick2 and P Gros1 Department of Biochemistry, McGill University, Montréal, Québec, Canada; 2Biotechnology Research Institute, Montréal, Québec, Canada 1 Systemic candidiasis is a significant cause of nosocomial infections and the mechanisms of defense against Candida albicans in humans remain poorly understood. Studies in animal models have demonstrated the importance of innate immunity in controlling the response to infection. Although Th1 cytokines have been shown to direct the overall outcome of infection, the precise role of the Th1/Th2 response and, more generally, the adaptive immune response as a whole, in systemic candidiasis, appears to apply mainly to the development of resistance to reinfection. A genetic approach to the identification of host factors regulating pathogenesis and susceptibility to C. albicans infection has been used in humans and in mouse models of infection. Mouse mutants bearing experimentally induced mutations in specific genes have provided a systematic tool for directly assessing the role of individual proteins in C. albicans susceptibility. Inbred mouse strains have been valuable in showcasing the spectrum of naturally occurring variations in initial susceptibility to infection, and type of disease developed. Crosses between resistant and susceptible strains have led to the detection of additional gene effects affecting innate immunity. Of particular interest is the major effect of a naturally occurring loss-of-function mutation in the C5 complement component that has become fixed in many inbred strains. These and other studies have shown that both a functional complement pathway and robust inflammatory response are critical for resistance to C. albicans. Genes and Immunity (2004) 5, 576–587. doi:10.1038/sj.gene.6364130 Published online 16 September 2004 Keywords: candida albicans; infection; genetics Candida albicans infections in humans Candida albicans is a fungus that can exist as a harmless colonizer or as an opportunistic pathogen, depending on the status of its host. In the majority of humans, Candida is a component of the residential microbial flora, where it exists as a commensal in the gastrointestinal and genitourinary tracts.1 However, in some— often immunocompromised—hosts, it has the capacity to colonize various tissues and organs, causing a range of diseases. It is the fungal species most frequently isolated in hospital-acquired infections2 and incidences of infection have risen with the increased prevalence of immunosuppressive therapies and broad-spectrum antibiotic use.3 C. albicans is a member of the genus Candida, and is the most frequent causative agent of candidiasis in humans.3 It is a diploid organism that undergoes mitosis and has a cell cycle with stages similar to other yeasts. It has only recently been shown that C. albicans undergoes mating, abolishing the previous view of Candida as an asexual organism.4,5 It exists in severally phenotypically distinct Correspondence: Dr P Gros, Department of Biochemistry, 3655 Sir William Osler Promenade, Room 907, Montreal, Quebec, Canada H3G1Y6. E-mail: [email protected] Received 22 June 2004; accepted 14 July 2004; published online 16 September 2004 cellular forms, including yeasts or blastospores, pseudohyphae, and hyphae. The dimorphic switch from yeast to hypha is thought to be one of the principal virulence mechanisms used by C. albicans to elude the host immune system during the switch from commensal to pathogen.6 Mutant strains of C. albicans that are unable to form either hyphae or yeasts are generally less virulent than wild-type strains.7–9 There are a number of additional properties of C. albicans that are thought to be important in the transition from benign colonizer to pathogen. The production of adhesion factors is important in the early stages of colonization and invasion of host tissues.10 Hydrolytic enzyme secretion, including the secreted aspartyl proteinase (Sap) family, phospholipase B enzymes, and lipases, have been demonstrated to have various roles in the infection process. These enzymes may be required for nutrient acquisition, distortion of host cell membranes to assist adhesion and tissue damage, and digestion of components of the host immune system to divert antimicrobial attack.11 All studies of putative virulence factors in C. albicans must be analyzed in the context of the immune status of the host, since in the absence of a compromised immune state, even the so-called virulent forms are not likely to cause infection. Interestingly, C. albicans has the ability to exploit a range of weakness in the host defense system, as illustrated by the spectrum of infections observed in humans. Obtaining a better understanding of what these weaknesses are can, in turn, provide insight into the host Innate immunity in resistance to Candida albicans A Tuite et al defenses that are required to protect against invasive infections by C. albicans. Types of candidiasis Infections caused by C. albicans can be broadly classified into two major categories: superficial and invasive candidiasis, with the major differences between these classes being the type of immune response elicited and the nature of the ensuing pathology. Superficial candidiasis encompasses a range of infections, including thrush, chronic atrophic stomatitis, chronic mucocutaneous candidiasis, and vulvovaginitis, and involve colonization of the skin and mucosal surfaces.12 These infections tend to be quite specific and self-limited in non-immunocompromised hosts, with the remedy involving local treatment and basic hygiene measures.12 Tlymphocyte-mediated immunity is the major player in the control of superficial infections. Among the forms of superficial candidiasis, chronic mucocutaneous candidiasis (CMC) is the syndrome with the strongest evidence for the involvement of host genetic factors in disease manifestation and outcome.13 CMC is characterized by persistent and recurrent infections of the skin, nails, and mucous membranes by Candida spp., especially C. albicans, and tends to be limited to patients with impaired T-cell function. CMC is often accompanied by endocrine and inflammatory disorders, and distinct syndromes have been identified based on the disorders that occur in conjunction with CMC.14 The AIRE (autoimmune regulator) gene has been associated with the autosomal recessive syndrome autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED).15,16 This gene was identified by positional cloning after mapping to chromosome 21q by linkage analyses in 14 Finnish families17 and subsequent confirmation in an Iranian Jewish population, giving a maximum LOD score of 10.2.18 An additional gene effect was identified in a large consanguineous family with CMC associated with thyroid disease that segregated as an autosomal dominant trait. A whole genome scan, followed by fine-mapping in 34 family members identified a 15-cM region on chromosome 2p with a maximum LOD score of 3.8.19 In contrast to cutaneous infections, which are heavily dependent on acquired immunity, innate immunity appears to be more important in determining disease outcome during invasive candidiasis. This dichotomy between the two major classes of infections is perhaps best exemplified by the prevalence of oropharyngeal candidiasis in HIV-positive and AIDS patients, who have suppressed T-cell function compared to the relative dearth of invasive infections in the same patients, who have relatively intact phagocytic function.20 Invasive candidiasis refers to Candida infections that occur at sites other than the skin or mucous membranes, with most cases caused by bloodstream invasion and subsequent spread of the organisms.21 These infections occur most frequently in immunocompromised hosts, although invasive candidiasis in critically ill, nonimmunosuppressed patients is becoming a growing concern.12 Chronic disseminated candidiasis (CDC) or hepatosplenic candidiasis occurs in patients undergoing intensive therapy for acute leukemia, where various aspects of their treatment put them at increased risk for infection.22 Patients develop signs of invasive candidiasis in multiple organs over a period of several weeks to months.21 Choi et al22 have identified a common haplotype in the promoter region of IL-4 that is overrepresented among acute leukemia patients with CDC, which they propose contributes to increased susceptibility to infection due to decreased IL-4 production. This haplotype was identified in a case/control study. Patients with the haplotype 1098T/589C/33C are at a greater risk of developing CDC, with an odds ratio of 2.16. Similarly, the protective 589T polymorphism is under-represented among CDC patients and this polymorphism is functional for enhanced IL-4 transcriptional activity.23 Disseminated or systemic candidiasis, another form of invasive infection, is characterized by the hematogenous spread of Candida to one or multiple organs.21 Systemic candidiasis is a life-threatening infection, with the overall prognosis comparable to septic shock with multiple organ failure.12 Although it is well established that systemic infection is associated with insufficiencies in host defenses,3 to date, no specific genes have been shown to cause a predisposition to infection. However, there is some anecdotal evidence that patients with congenital defects affecting phagocyte function, such as myeloperoxidase (MPO) deficiency, have a higher occurrence of systemic candidiasis than healthy individuals.24 Among the major risk factors for candidiasis are: neutropenia, broad-spectrum antimicrobial treatment, damage to oropharyngeal mucosa through use of cytostatic drugs, use of central venous catheters, parenteral nutrition, and breaches of skin integrity due to injury or burns.1,3 577 Epidemiology The incidence of hospital-acquired infections caused by Candida has increased steadily since the 1970s; this is likely due to both an increase in the patients who are rendered susceptible to infection due to immunosuppressive procedures and improved clinical procedures to identify yeasts as the causative agents of nosocomial infections.25 Candidiasis is the fourth most common cause of nosocomial bloodstream infections.1 Although invasive candidiasis occurs in 1–8 percent of patients admitted to hospitals, its occurrence is higher in intensive care units, with infection rates of approximately 10 percent.12 In intensive care units, candidiasis is accountable for up to 15 percent of all nosocomial infections.12 The mortality attributed with candidemia has been reported to range from 25 to 60 percent.26 There is also a considerable morbidity associated with Candida infections, with a median 30 day increase in hospital stay seen in nonfatal cases compared to uninfected controls.27 Thus, although the incidence of candidiasis in the general population is not a major problem, the cost in terms of attributable morbidity and mortality among hospitalized patients is significant. Disease pathology Systemic candidiasis is of particular concern due to the severity of these infections in already hospitalized, and often critically ill, patients and the difficultly of treatment Genes and Immunity Innate immunity in resistance to Candida albicans A Tuite et al 578 with antifungal agents. Although disease manifestation varies with respect to the target organs and severity of colonization, some generalizations about infection initiation, progression, and outcome can be made. Systemic candidiasis usually begins with the penetration of mucosal barriers, giving the pathogen access to the bloodstream, where it is subsequently disseminated throughout the body,26 although external introduction through central venous catheters and other surgically implanted devices has also been reported.28,29 Candida lesions have been found in virtually every tissue of the body, although the major targets of infection are the digestive tract, lungs, kidney, and brain.30 The heart is colonized nearly as often, while the liver and spleen are less frequently infected. Emboli with yeasts and pseudohyphae are often found in the blood vessels of systemic candidiasis patients. C. albicans causes the formation of small, white lesions in the parenchyma of deep organs.30 These lesions are multiple discrete abscesses or microabscesses that superficially resemble those formed in miliary tuberculosis or by diffuse cancer metastases. Candida yeasts and filamentous forms tend to be profuse within the abscesses, and are usually, but not always, associated with neutrophil infiltrates. The study of systemic candidiasis in humans, especially concerning factors which predispose to infection, has provided some insight into the contribution of the host in regulating disease susceptibility. It is clear that the innate immune response, especially neutrophilmediated phagocytosis or killing, is of particular importance in preventing systemic candidiasis. However, in the absence of an obvious genetic link to susceptibility to candidiasis, further elucidation of the contribution of the host in determining disease susceptibility, progression, and outcome has necessitated the use of animal models. Animal models Animal models have been used extensively as a tool to better understand systemic C. albicans infections, from the perspective of both the host and the pathogen. Although the mouse is by far the most commonly used animal rats, rabbits, and guinea-pigs are all approximately equally as susceptible to infection as the mouse and display similar disease pathology.30 Systemic infections in these animals resemble human candidiasis, with the kidney being the major target of infection.31 The major routes used to establish infection are intravenous (i.v.) and intraperitoneal (i.p.) injection. I.v. inoculation leads to the establishment of an acute systemic infection involving multiple deep organs. I.p. infection also leads to disseminated infection, but the required inoculum to establish a pathology similar to that observed in i.v. infection is approximately 10-fold higher.30 An alternate model uses gastrointestinal (g.i.) candidiasis as a model for systemic candidiasis of endogenous origin, but establishing an infection using this method is more variable, since it relies on dissemination of Candida from the g.i. tract into the bloodstream, prior to organ colonization. An extensive review of the progression of systemic candidiasis in the mouse has been conducted by Odds.30 In brief, systemic infections in the mouse are characterized by a rapid clearance from the blood, with the Genes and Immunity highest fungal loads isolated from the lungs and liver in the first hours after C. albicans injection. By 1 to 2 days postinfection, the kidneys have the highest fungal burden, and this increases with time, due to active replication. In contrast, fungal loads in the lung, liver, and spleen decline over time. Depending on the dose and strain of mouse used, animals may begin dying from overwhelming infection within the first 24 h, presumably from kidney failure caused by acute pyelonephritis.31 After the first week of infection, the kidney is the most obviously affected organ in all surviving animals. Its surface is studded with macroscopic white abscesses. These abscesses are focal collections of Candida yeast, hyphae, and pseudohyphae. The appearance of lesions varies with the strain of mouse used. They can show little evidence of an inflammatory reaction, contain large infiltrates of polymorphs and/or macrophages, or form granulomas. A similar pattern of tissue damage has been observed in other affected organs, although the extent and number of lesions tends to be reduced, compared to the kidney. Phenotypes measured Doses and strains of C. albicans used to study susceptibility in the mouse tend to vary across experiments, with inocula ranging from 103 to 108 yeasts having been tested.30 The LD50 for most C. albicans isolates tested in immunocompetent mice is between 104 and 106 yeasts. Both the strain of C. albicans and the growth conditions used have been shown to influence overall lethality during infection.32 The three phenotypes most frequently measured to ascertain host response to C. albicans infection are tissue damage, fungal burden, and mortality, with each representing a different aspect of the host– fungus interaction.33 Tissue damage is assessed microscopically and is a somewhat difficult phenotype to measure quantitatively. It represents the interaction between Candida and the host effector cells at a given point in time and gives an idea of the type of inflammatory response elicited and the major cell types recruited during this response. In the mouse, the organs most involved in disease pathology are the kidney, brain, and heart. Measurements of total fungal burden in affected organs at a given point in time give a more quantitative measure of infection and reflect the candidacidal or candidastatic efficiency of the host response. Mortality indicates the overall susceptibility to infection. A correlation between kidney fungal load and mortality has been observed.34–36 Cytokine levels at various time points throughout infection provide an additional measure of the differences in the host response to systemic candidiasis. Early in infection, susceptible mice have elevated levels of TNF-a and IL-637 (Mullick et al, in press) and these cytokines are useful predictors of disease outcome. Similarly, mRNA levels of IFN-g, TNF-a, and IL-6 in the brains of susceptible mice with systemic candidiasis were found to correlate with the total infectious burden.38 Mouse models of systemic candidiasis provide a wellestablished foundation upon which to base studies into the mechanisms of the host response to infection. Patterns of tissue damage in targeted organs have been described, as has overall disease pathology and progression. Selection of phenotypes that show clear differences between resistant and susceptible animals enables the Innate immunity in resistance to Candida albicans A Tuite et al characterization of the effects of various cellular and gene deficiencies on susceptibility. As a result, mouse models have been used with great success to elucidate the involvement of various cell populations in the defense against systemic infection. Effector cells In an attempt to characterize the contribution of specific cell types of the immune system, the response to infection has been studied in immunodeficient strains, which have defects or deficiencies in one or more cell populations. In addition, the specific depletion of a given population through antibody neutralization or chemical means has been used. Neutrophils are the major effector cells early in systemic infection. They are rapidly recruited to the sites of infection. Among the mechanisms employed by neutrophils to control infection are: phagocytosis of C. albicans,39,40 production of reactive oxygen species,41 and the release of mediators, including cytokines and antimicrobial substances.42 In animals that are pretreated with agents to reduce the number of neutrophils, there is a significantly increased susceptibility to systemic C. albicans challenge.30,43 Similarly, in mice that are selenium deficient, a condition where the candidacidal activity of neutrophils is impaired, there is a high susceptibility to candidiasis.44 Beige mice, which have impaired natural killer (NK) cell and neutrophil function, have a reduced ability to kill C. albicans blastospores and hyphae compared to immunocompetent controls.45,46 On average, two yeasts are ingested per neutrophil, although up to ten yeasts have been observed per cell.47,48 The proportion of ingested yeasts that are killed is between 20 and 30 percent, regardless of the number ingested, with unkilled yeasts capable of germinating and growing out through the cell wall.49 Neutrophils kill hyphal and yeast forms of Candida at similar rates, although yeasts tend to be killed intracellularly and hyphae are predominantly killed extracellularly.39,40 In addition to phagocytosis and killing, neutrophils are capable of directing the development of the host immune response through cytokine secretion.42 More contentious is the role of macrophages in the host response against C. albicans. In vitro studies have shown that the candidacidal capacity of macrophages is worse, similar to, or better than neutrophils, depending on the experimental conditions.50 One study showed that mouse peripheral blood leukocytes and peritoneal macrophages ingest live C. albicans at the same rate, but leukocytes more effectively killed intracellular yeasts.37 Nramp1 is a divalent cation transporter at the phagosomal membrane of macrophages and neutrophils, with mutations in this protein causing an increased susceptibility to several intracellular pathogens.51 A role for Nramp1 in the killing of Candida yeasts and hyphae has been shown, despite the fact that they are killed by intracellular and extracellular mechanisms, respectively.52 Interestingly, NRAMP1 is upregulated in neutrophils and is recruited to the membrane of C. albicanscontaining phagosomes.53 To date, no in vivo studies in mice have unambiguously demonstrated an absolute requirement for macrophages in the resolution of C. albicans infections.43,54 Depletion of NK cells was found to have no effect on resistance to primary C. albicans infections or in conferring protection against reinfection55 and it is generally accepted that NK cells do not play an essential role in host response against candidiasis.55,56 A role has been proposed for dendritic cells (DCs) as initiators of adaptive immunity. They are capable of phagocytosing both the yeast and hyphal forms of C. albicans, albeit by different mechanisms.57 Depending on the form of Candida engulfed, different downstream cellular events are initiated. Since DCs initiate responses in naı̈ve T cells and participate in T helper (Th) cell education, they are expected to have a role in regulating the response to infection.58 Although the relative contribution of DCs to the control of systemic candidiasis has not yet been clearly demonstrated, it is believed that detection by DCs through Toll-like receptors (TLRs) is involved in pathogen discrimination and initiation of the appropriate adaptive immune response.59,60 The involvement of T- and B-lymphocytes in host defense against systemic candidiasis has been studied in nude (thymus deficient)60–62 and SCID (absence of T and B cells)43,63 mice. These cell populations are not essential for protection against primary infection and, in some instances, deficient strains are actually more resistant.43,47,63,64 Similarly, thymectomized, irradiated, and bone marrow reconstituted (TXB) mice showed no difference in disease outcome when compared to immunocompetent controls.65 However, TXB mice developed no resistance to rechallenge. Even at the later stages of infection, when acquired immunity is expected to be operative (days 10–21), no demonstrable role was observed for T or B cells.63 The major role of T cells in protection against systemic candidiasis appears to be in the development of resistance to reinfection after initial inoculation with a vaccine strain of C. albicans.36,65 CBA/N mice have X-chromosome-linked immune deficiency (XID), which manifests primarily as a defect in humoral immunity, specifically a reduced serum antibody production. These mice have been used as a B-cell defective mouse model.66 In both primary and secondary infections, no difference was seen between CBA/N mice and B-cell-competent CBA/J mice in response to i.v. C. albicans infection.66 Conversely, Wagner et al67 observed that JHD B-cell knockout mice, which are generated by the targeted disruption of the gene segment that encodes the joining region of the Ig H chain,68 were more susceptible to systemic infection than wild-type controls. Although antibodies directed against C. albicans are produced upon exposure to the pathogen, the presence of both protective and nonprotective antibodies complicates any attempt to determine the overall importance of humoral immune mechanisms in conferring protection.69 Efforts to determine the relative contributions of various cells of the immune system to the host response to infection have benefited from the study of immunodeficient mouse models. It is clear that neutrophils are the major effector cells involved in controlling the spread and replication of C. albicans during infection. However, it is likely that other phagocytic cells, such as macrophages are also important in the host response, although a redundancy in their function may preclude their detection in depletion studies. Less obvious is the role of T and B cells in infection resolution, with most studies suggesting that these cell populations are relatively dispensable in primary infection resolution. 579 Genes and Immunity Innate immunity in resistance to Candida albicans A Tuite et al 580 Knockout mice have been used to further clarify the role of both the innate and acquired arms of the immune system in progression of systemic candidiasis (Table 1). The effect of a deficiency of a specific component of the immune system provides a more refined view of the pathways and networks activated by the host in response to infectious challenge. Deficiencies of innate immunity and involvement in early infection Innate immunity is a major component of the response to systemic C. albicans infection, since it is the first arm of the immune system to detect and react to the invading pathogen. C. albicans is cleared rapidly from the bloodstream and becomes lodged in various organs. Among the first cells at the site of infection are professional phagocytes, and deficiencies that affect the ability of these cells to ingest and kill C. albicans are implicated in increased susceptibility to infection. Detection of Candida occurs mainly through TLRs and the complement system. Uptake of unopsonized C. albicans occurs through pattern recognition receptors,70 whereas serum-opsonized Candida is phagocytosed by complement receptors on phagocytic cells.49 TLRs have the ability to recognize the invading fungi and initiate signaling cascades to recruit and activate effector cell populations.70 TLR2 and TLR4 are the most well characterized of the TLRs with respect to their involvement in fungal recognition. TLR4, which is expressed on the surface of macrophages, DCs, and endothelial cells,71,72 recognizes C. albicans-derived mannan,73 possibly in conjunction with the CD14 receptor. It is involved in the control of fungal replication in the kidneys, likely by inducing the secretion of CXC cytokines such as KC and MIP-2, which subsequently recruit neutrophils to the site of infection.74 Conflicting results exist for the mechanism of TLR2’s involvement in the regulation of infection outcome. TLR2 expression is detected on neutrophils, DCs, and monocytes,72 and it is thought to detect phospholipomannan from the cell-wall surface of C. albicans.75 Villamon et al76 have proposed that TLR2 is essential in murine defense against C. albicans infection, and in the absence of TLR2, mice have a decreased survival time after infection. Conversely, Netea et al77 showed that signaling through TLR2 appears to be detrimental to host survival, since the Table 1 Involvement of genes in resistance to primary infection with C. albicans Gene Phenotype measured Fungal load Mortality Fungal load Mannose receptor Mortality and NADPH oxidase Mortality and MPO Mortality and TNF-a Mortality and IL-6 Mortality and IFN-g Fungal load IL-12 Mortality and IL-10 Fungal load IL-4 Fungal load Role in primary infection TLR4 TLR2 Genes and Immunity fungal fungal fungal fungal fungal load load load load load fungal load kSusceptibility Uncertain mSusceptibility No effect kSusceptibility kSusceptibility kSusceptibility kSusceptibility kSusceptibility No effect mSusceptibility kSusceptibility absence of TLR2-mediated signaling results in an increased resistance to systemic candidiasis. They attribute this to an expansion of the CD4 þ CD25 þ regulatory T-cell population and a decreased macrophage candidacidal capacity in the presence of TLR2. These findings were supported by Bellocchio et al,78 who found a reduced fungal load in the kidneys of TLR2-deficient mice but no difference in mortality. The reason for the apparently opposing role of TLR2 in controlling the response to systemic candidiasis is unclear, but may reflect differences in the strain of C. albicans used, since TLRs have been shown to discriminate between fungal morphotypes.32,78 The mannose receptor is another pattern recognition receptor that was thought to recognize C. albicans through the binding of specific cell wall glycans, based on in vitro studies.79,80 However, a recent study in mannose receptor knockout mice showed no significant difference in overall susceptibility compared to wildtype controls,81 suggesting that this is not a major receptor for C. albicans detection, or that a redundancy exists with the TLR4/CD14 system. The complement system, which is composed of plasma proteins and membrane proteins, is important in the opsonization, phagocytosis, and killing of microorganisms.82 C. albicans activates the complement system through the alternative pathway.83 Guinea-pigs deficient in complement component 4 were unaffected in their ability to respond to infectious challenge with C. albicans, suggesting that the classical pathway is not essential in pathogen recognition and clearance.83 The involvement of the membrane attack complex in direct killing of the pathogen has not been demonstrated.84 The chemotactic activity of the complement is likely to be important in recruiting effector cells to infected tissues. Complement enhances phagocytosis of C. albicans by murine blood leukocytes, but it does not affect killing by these cells.85 MPO and NADPH oxidase generate reactive oxygen species, which are an integral part of the microbicidal system used by neutrophils against Candida.86 Defects in these complexes have been implicated in enhanced susceptibility to candidiasis.24,41 NADPH oxidase generates superoxide anion during the phagocytosis-associated respiratory burst. Mice with X-linked chronic granulomatous disease (X-CGD), which have deficiencies in NADPH oxidase, have an increased susceptibility to disseminated candidiasis, as determined by survival time and fungal load.86 This correlates with the observed decreased in vitro candidacidal activity of neutrophils from human CGD patients, despite the normal uptake of C. albicans.49 MPO is mainly expressed in neutrophils and monocytes and leads to the production of hypochlorous acid.87 In vivo studies in MPO knockout mice demonstrated that these mice are increasingly susceptible to systemic candidiasis,86,87 in agreement with the finding in humans of an elevated rate of C. albicans infections in patients with congenital MPO deficiencies.24 Neutrophils isolated from a patient with disseminated candidiasis were able to ingest but not kill C. albicans in vitro, and these leukocytes were subsequently found to be completely devoid of MPO.49 The innate immune system is a major player in the detection, uptake, and killing of C. albicans during systemic infection. Studies in knockout mice have Innate immunity in resistance to Candida albicans A Tuite et al revealed the importance of the TLR pathway and the complement cascade in the initial interaction between the host and pathogen. Following detection and possible phagocytosis, depending on the morphogenic form of Candida, the microbicidal system is key in controlling the replication and spread of the invading organism. Th1 and Th2 cytokines in experimental systemic candidiasis Although no definitive role for T cells has been observed during infection, it is clear that cytokine production by Th cells can influence infection outcome. The Th cell response during systemic candidiasis in the mouse has been well studied.88 The profile of Th cell differentiation in response to candidiasis is directed by cytokines produced during the initial phase of infection. Various effector cells have been shown to secrete cytokines, thereby influencing the adaptive immune response by directing CD4 þ Th cell differentiation. These cytokines prime the activation of naı̈ve T cells into either Th1 or Th2 cells. The Th1 response is associated with cellular immunity, leading to the activation of macrophages and neutrophils to a candidacidal state. The Th2 response is involved in the development of humoral immunity, resulting in the activation of B cells. Since the cytokines produced by one T-cell subset tend to inhibit activation of the other set, the overall effect is the dominance of either humoral or cell-mediated immunity, although mixed Th1/Th2 responses do occur.89 In the case of systemic candidiasis, the development of a Th1 response is associated with protection against infection, while a Th2 response is more frequently observed in susceptible hosts.90 The overall importance of the Th1/Th2 paradigm in disease outcome is mainly applicable to the development of protective immunity after initial infection. The development of protective immunity has been mainly studied by evaluating the effects of various cytokine deficiencies on resistance to reinfection, following primary infection with an avirulent vaccine strain.36 In this system, Th1 cytokines have been shown to be important in protective immunity development, while Th2 cytokines tend to prevent the development of resistance to reinfection.57 The roles of these various cytokines in primary infection have also been studied, and individual cytokines that can be classified as either Th1 or Th2 do influence disease progression and outcome. Thus, they likely have a dual role during infection, regulating both effector cell functions and T-cell development. The proinflammatory cytokine TNF-a is important in innate and adaptive immunity against C. albicans. It recruits and activates effector phagocytes, induces costimulatory molecules, and is required for Th1 response induction.90 TNF-a production is initiated in the first few hours after infection, primarily in monocytes and macrophages,91 although neutrophils also secrete this cytokine.92 It does not appear to be a mediator of early death, since neutralization does not alter the time of death, and administration of TNF-a modestly protects against infection.93 TNF-a knockout mice have a decreased survival time and higher fungal loads in the kidney, spleen, and liver.94 In the absence of TNF-a, there is a reduced recruitment of neutrophils to the sites of infection, which likely explains the increased susceptibility of knockout mice.95 Similar findings are seen in IL6 knockout mice,96 with IL-6 deficiency leading to a more severe pathology. These cytokines act very early in infection, arguing against Th1 response induction as their primary role during infection. Since the TNF-a and IL-6 knockout mice die early in infection, even upon injection of a low virulence strain of C. albicans, their effect on the development of protective immunity has not been determined.36 However, the in vitro analysis of cytokine production of activated spenocytes from infected mice showed the high levels of production of Th2 cytokines.96,97 IFN-g is another Th1 cytokine that is important in primary infection. IFN-g knockout mice are more susceptible than wild-type controls to systemic candidiasis.98 Experiments investigating the roles of IL-12 and IFN-g in systemic infection99 highlight the complexity of the balance of cytokines during infection progression. Despite its importance in protection against reinfection,100 native IL-12 is not required during primary systemic candidiasis,36,101 and exogenous IL-12 administration actually exacerbates disease severity, apparently by inducing IFN-g overproduction. The IFN-g receptor is required for protection against reinfection with C. albicans.102 The Th2 cytokines IL-10 and IL-4 have disparate effects on the control of infection progression. IL-10 inhibits cytokine production by Th1 cells, and has a detrimental effect on resistance to systemic infection, as demonstrated by both neutralization103 and knockout studies.104 In contrast, IL-4 appears to be important in protection against the early stages of fungal infection, with knockout mice having an increase in fungal replication in target organs.104 Interestingly, this Th2 cytokine is required for the development of resistance to re-infection.105 Based on these and other studies on the effects of various cytokines on disease outcome, it is apparent that they are an integral part of the host response to candidiasis. However, the relatively simplistic view of the protective Th1 vs the nonprotective Th2 response, while providing a framework by which to better understand the nature of the response to infection, is not able to fully encapsulate the complexity of this response. In particular, the timing of cytokine production can have a major influence on the effect on infection progression. A number of protective Th1 cytokines have their effects very early during infection, before acquired immunity is active. However, in the context of protection from reinfection, Th1 response development is important. Numerous studies in knockout mice have clarified the importance of individual genes in disease pathogenesis. However, these analyses are limited to the exploration pathways and molecules that are predicted to be relevant to the host defense against invading pathogens. Additionally, these studies in mice may be confounded by unforeseen genetic background effects. A parallel approach to identify novel or not previously implicated mechanisms of host defense is gene discovery through the use of inbred mouse strains. 581 Studies in inbred mouse strains From studies in knockout mice, it is clear that the host response to candidiasis is complex, involving a number host–pathogen interactions, as well as interactions between various components of the immune system. An alternative approach to identifying host genetic Genes and Immunity Innate immunity in resistance to Candida albicans A Tuite et al 582 factors involved in disease outcome is through the use of inbred mouse strains, which possess a diversity of genetic backgrounds that have been fixed through generations of inbreeding. Over the years, the response of a number of different inbred strains to systemic candidiasis has been assessed, with fungal load and mortality being the two most commonly measured phenotypes (Table 2).33,35,74,106–111 For the more frequently tested strains, results between experiments are fairly consistent, despite differences in experimental conditions, such as C. albicans strain and dose used and the time of assessment of fungal load (fixed time point or at time of death). Interstrain differences are apparent with respect to susceptibility to infection with C. albicans, although correspondence between mortality and fungal burden is not always seen, suggesting the possibility of alternate genetic control for these traits. Exploitation of differences between strains may allow for the mapping of loci that affect the host response to infection, which in turn may lead to the identification of their counterparts in humans. Eventual cloning of any identified genes would provide insight into factors affecting susceptibility to systemic infection and potentially improve treatment of these infections. Carg1 and Carg2 To date, two gene effects controlling susceptibility to systemic candidiasis, as measured by tissue damage, have been proposed. The discrete differences in tissue damage seen in BALB/c and CBA/CaH and the segregation pattern observed in a [BALB/c CBA/ CaH] F2 population led Ashman and Papadimitriou112 to propose the existence of a codominant, Mendelian gene. The resistance conferred by this gene appeared to be acting through cells derived from the bone marrow. They subsequently investigated this trait in AKR and C57/L mice; the kidneys of AKR mice showed moderate tissue damage after i.v. infection, whereas the damage in C57/L kidneys was of mild severity.113 Using a panel of 15 AKXL recombinant inbred strains, this gene, C. albicans resistance gene 1 (Carg1), was tentatively assigned to chromosome 14.114 Carg1 is hypothesized to be involved in overall susceptibility to tissue damage. However, tissue damage in some of the recombinant inbred strains was more severe than either of the parental strains, suggesting that a second locus, designated C. albicans resistance gene 2 (Carg2), may be involved. This gene is proposed to influence specifically the level of susceptibility of the kidney to fungal damage,115 having relatively minor effects in overall susceptibility. The genomic location of this putative gene has not yet been mapped. The characterization of the roles of these loci in the mechanism of resistance to infection, as well as any interaction with other components of the host immune system, awaits further mapping and cloning. Involvement of complement component 5 In looking at the susceptibility to C. albicans across mouse strains, the importance of complement component 5 (Hc/C5) in determining disease outcome is striking. The association of C5 deficiency with increased susceptibility is clear, although the correlation is not perfect. A similar relationship is seen for susceptibility to infection with Cryptococcus neoformans116 and Saccharomyces cerevisiae.117 Up to 40 percent of the commonly used inbred mouse strains118 have a 2-bp deletion in an exon near the 50 -end of the mRNA, introducing a premature stop codon 4-bp downstream of the deletion.119 This leads to the production of a truncated 216 amino-acid translation product compared to the wild-type 1680 amino-acid protein. This truncated protein is not secreted.119 The functional consequences of a C5 deficiency in the response to systemic candidiasis have been studied extensively.111,120,121 In two histocompatible B10.D2 congenic strains that are expected to differ primarily in their C5 status, the C5-deficient strain had a higher fungal burden Table 2 Response of inbred strains to systemic infection with C. albicans Strain A/J AKR/J B10.D2/oSn B10.D2/nSn BALB/c BALB/cN C3H/HeN C3H/HeJ C57/L C57BL/10SnJ C57BL/6J C57BL/6J beige-J C57BL/KsJ CBA/CaH CBA/J CBA/N DBA/1 DBA/2 NZB RF/J SJL a Hc allelea 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 — C5 deficient; 1 — C5 sufficient. Genes and Immunity Survival Fungal load Susceptible Resistant — — Resistant Susceptible Resistant Susceptible — — Resistant Intermediate Resistant Susceptible/Intermediate — Intermediate Resistant Susceptible Susceptible Susceptible Resistant Susceptible Susceptible/Intermediate Susceptible Resistant Resistant Resistant Resistant Susceptible/Resistant Resistant Resistant Resistant Intermediate Resistant Intermediate Susceptible Susceptible Resistant Susceptible — — — References 33, 107, 110 33, 35, 107 111 111 33 106 33, 74 74, 106, 108, 110, 111 33 35 33, 106, 107, 110 106 106,107 33, 107 35, 110 106 33, 107 33, 107, 110 109 35 107 Innate immunity in resistance to Candida albicans A Tuite et al in the kidney early in infection.111 Although the presence of C5 did allow for a more rapid initial control of C. albicans replication after challenge, as the infection proceeded there was no significant difference between the C5-sufficient and -deficient strains in terms of kidney fungal load. This suggests that additional genetic factors can contribute to the later stages of infection control, even in the absence of functional C5. The treatment of C5-deficient DBA/2 mice, which are extremely sensitive to infection, with C5-containing serum had a protective effect, reducing fungal load, and increasing survival time.120 Depletion of neutrophils did not completely abrogate the protective effects of C5 treatment, indicating that C5 is not acting exclusively through neutrophils and supporting the view that other cell populations are also recruited to control C. albicans replication. We have provided additional evidence for the important role of C5 in regulating the response to infection. The recombinant congenic strain BcA70, which derives 87.5 percent of its genome from the resistant, C5-sufficient C57BL/6J (B6) strain and 12.5 percent from the susceptible, C5deficient A/J strain,122 is as susceptible to infection as A/ J. Despite being on a resistant background, BcA70 is deficient for C5, suggesting that C5 is a major determinant in controlling the response to systemic candidiasis. To map genes controlling the host response to systemic candidiasis, we have assessed fungal load in the kidney and brain in a susceptible and a resistant inbred strain. Fungal load in the brain is significantly correlated with kidney fungal load (Po0.0001), suggesting that they are under the same genetic control. Based on fungal loads in these organs measured 48 h after i.v. infection, A/J is susceptible and B6 is resistant to infection. The distribution pattern for fungal load measured in [A/J B6] F2 progeny was indicative of susceptibility acting as an autosomal recessive trait under simple genetic control. A genome-wide scan on the DNA from 128 F2 mice followed by linkage analysis led to the identification of a statistically significant linkage on the proximal portion of chromosome 2, which we determined to be due to the effect of C5. We also looked at heart fungal load, serum TNF-a levels, and overall mortality in additional F2 mice. Segregation analysis verified that these differences between susceptible and resistant mice were explained by the effect of C5. Thus, C5 deficiency in mice is associated with a decreased resistance to infection. This gene acts in a recessive manner, with heterozygotes displaying a similar degree of resistance as their wildtype counterparts. Our results provide further support for the importance of C5 in regulating the response to systemic candidiasis (Tuite, Mullick, and Gros, unpublished). We have observed that C5 deficiency leads to elevated serum TNF-a levels and Ashman and Papadimitriou37 have shown that TNF-a secretion is proportional to the infectious burden. A clue as to the role of C5 in controlling TNF-a production is provided by experimental studies of sepsis; in a rat model, C5a has been shown to inhibit TNF-a production in neutrophils through induction of IkBa, preventing nuclear translocation of NFkB and thereby leading to reduced transcription of TNF-a.123 This mechanism is specific for neutrophils and, in fact, the opposite effect is observed in macrophages, with C5 enhancing TNF-a production. Thus, the increased serum TNF-a levels in C5-deficient mice may be due to uncontrolled TNF-a production in circulating neutrophils induced in response to the pathogen. The precise role of C5 in disease pathogenesis is likely due to C5a, which is generated by cleavage of C5 during complement system activation.124 The other cleavage product, C5b, has a role in opsonization and is required for assembly of the membrane attack complex, but its role in control of candidiasis is not clear. Specifically, direct lysis by the membrane attack complex does not appear to be an essential mechanism for the killing of C. albicans in vivo.84 Although complement does accelerate the rate of C. albicans phagocytosis by neutrophils,49,85 the fact that Candida is cleared equally as rapidly from the bloodstream of C5-sufficient and -deficient mice argues against the opsonization activity of C5b as regulating the efficiency of the host response. C5a is an anaphylatoxin and a chemoattractant.125 As an anaphylatoxin, C5a induces various features of acute inflammation, including increased blood flow, edema, and smooth muscle contraction.126 However, the profile of cytokines produced in A/J mice during infection is characteristic of an extreme inflammatory reaction suggesting that in the absence of C5, there is actually an unregulated production of proinflammatory molecules. Contrary to C5’s more well-characterized role as an inducer of inflammation, in candidiasis C5 may have a role as a negative regulator of the inflammatory response. The most defined role for C5a during invasive candidiasis is as a chemotactic agent for neutrophils. In the absence of C5a, mice have been shown to have a reduced recruitment of effector cells to the site of infection. Neutrophils express high levels of the C5a receptor (C5aR/CD88),126 explaining their rapid infiltration to sites of infection in response to C5. C5a signaling pathways in neutrophils lead to the assembly of catalytically-active NADPH oxidase at the cell membrane, which has an established role in the killing of phagocytosed C. albicans (discussed above). C5’s role as a mediator of host response to infection has been demonstrated in a number of organisms and against a number of pathogens. In particular, C5 deficiencies in humans are associated with recurrent Neisserial infections.82 In mice, C5 deficiency has been linked to susceptibility to Listeria monocytogenes infection.127 Additionally, virulent group A streptococci have been shown to express a cell-surface factor capable of inactivating C5a, which they use as a mechanism of eluding the host immune response.128 The results of the genome-wide scan clearly illustrate the importance of C5 as a major gene controlling the differential susceptibility to systemic C. albicans infections seen in A/J and B6 mice. A deficiency in C5 explains increased fungal outgrowth in target organs, elevated serum TNF-a levels, and an increase in overall mortality in susceptible mice. The physiological relevance of a C5 deficiency in humans with systemic candidiasis is unclear, since although this deficiency renders patients increasingly susceptible to a range of microbial infections, an increased incidence of candidiasis has not been reported.124 This may simply be due to the decreased pathogenicity of Candida, relative to other microorganisms that are better adapted at exploiting the weakened defense systems of a host that is deficient in C5. 583 Genes and Immunity Innate immunity in resistance to Candida albicans A Tuite et al 584 Conclusion Recognition of systemic candidiasis as an important medical concern has been increasing in concert with the rise in immunosuppressive and broad-spectrum antimicrobial therapies. The development of candidiasis is due to the complex interaction between the host and colonizing C. albicans, in addition to environmental factors. As resistance to antifungal agents becomes a growing concern, a better understanding of the host– pathogen interaction is invaluable for the development of any new therapies. Animal models provide a useful tool to study the response to systemic candidiasis and studies in mice have indicated the importance of the innate immune system in controlling infection. In particular, the involvement of neutrophils in the initial stages of infection parallels the importance of neutrophils in human infections and validates the mouse as a physiologically relevant model of systemic candidiasis. Acknowledgements PG is a distinguished scientist of the Canadian Institutes of Health Research and a James McGill Professor. AT is supported by a studentship from the Natural Sciences and Engineering Research Council of Canada. 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