Dendritic cells discriminate between yeasts and hyphae of the fungus Candida albicans. Implications for initiation of T helper cell immunity in vitro and in vivo.

The fungus Candida albicans behaves as a commensal as well as a true pathogen of areas highly enriched in dendritic cells, such as skin and mucosal surfaces. The ability of the fungus to reversibly switch between unicellular yeast to filamentous forms is thought to be important for virulence. However, whether it is the yeast or the hyphal form that is responsible for pathogenicity is still a matter of debate. Here we show the interaction, and consequences, of different forms of C. albicans with dendritic cells. Immature myeloid dendritic cells rapidly and efficiently phagocytosed both yeasts and hyphae of the fungus. Phagocytosis occurred through different phagocytic morphologies and receptors, resulting in phagosome formation. However, hyphae escaped the phagosome and were found lying free in the cytoplasm of the cells. In vitro, ingestion of yeasts activated dendritic cells for interleukin (IL)-12 production and priming of T helper type 1 (Th1) cells, whereas ingestion of hyphae inhibited IL-12 and Th1 priming, and induced IL-4 production. In vivo, generation of antifungal protective immunity was induced upon injection of dendritic cells ex vivo pulsed with Candida yeasts but not hyphae. The immunization capacity of yeast-pulsed dendritic cells was lost in the absence of IL-12, whereas that of hypha-pulsed dendritic cells was gained in the absence of IL-4. These results indicate that dendritic cells fulfill the requirement of a cell uniquely capable of sensing the two forms of C. albicans in terms of type of immune responses elicited. By the discriminative production of IL-12 and IL-4 in response to the nonvirulent and virulent forms of the fungus, dendritic cells appear to meet the challenge of Th priming and education in C. albicans saprophytism and infections.

NO-aspirin protects from T cell-mediated liver injury by inhibiting caspase-dependent processing of Th1-like cytokines.

BACKGROUND & AIMS: Concanavalin A (con A)-induced hepatitis is an immunomediated disease in which assembly of CD4(+) T cells and T helper (Th)1-like cytokines causes Fas-mediated liver cell death. Nitric oxide (NO) modulates Th1 response in vitro. NCX-4016 is an NO-aspirin derivative that spares the gastrointestinal tract and shares molecular targets with NO. The aim of this study was to investigate whether this NO-aspirin modulates Th1-like response induced by con A. METHODS: BALB/c mice were injected with 0.3 mg con A per mouse alone or in combination with NO-aspirin (18-100 mg/kg) or aspirin (10-55 mg/kg). RESULTS: NO-aspirin, but not aspirin, caused a dose-dependent protection against liver damage induced by con A. At a dose of 100 mg/kg, NO-aspirin caused a 40%-80% reduction of interleukin (IL)-1beta, IL-12, IL-18, interferon (IFN)-gamma, and tumor necrosis factor alpha production without affecting cytokine messenger RNA expression. NO-aspirin prevented Fas, Fas ligand, and IL-2 receptor up-regulation on spleen lymphocytes and Fas ligand on hepatocytes and caused the S-nitrosylation/inhibition of IL-1beta-converting enzyme-like cysteine proteases (caspases) involved in the processing and maturation of IL-1beta and IL-18. IL-18 immunoneutralization prevented IFN-gamma release and protected from liver injury induced by con A. In contrast to a selective caspase 1 inhibitor, zVAD.FMK, a pancaspase inhibitor, prevented IFN-gamma release and protected the liver from injury. CONCLUSIONS: Th1-like response induced by con A is mediated by IL-18 and requires activation of multiple caspases. NCX-4016 causes the S-nitrosylation/inhibition of caspases involved in cytokine production. Inhibition of Th1-like response is a new anti-inflammatory mechanism of action of NO-aspirin.

Antifungal type 1 responses are upregulated in IL-10-deficient mice.

C57BL/6 mice are highly resistant to infections caused by Candida albicans and Aspergillus fumigatus. To elucidate the role of IL-10 produced by C57BL/6 mice during these infections, parameters of infection and immunity to it were evaluated in IL-10-deficient and wild-type mice with disseminated or gastrointestinal candidiasis or invasive pulmonary aspergillosis. Unlike parasitic protozoan infection, C. albicans or A. fumigatus infection did not induce significant acute toxicity in IL-10-deficient mice, who, instead, showed reduced fungal burden and fungal-associated inflammatory responses. The increased resistance to infections as compared to wild-type mice was associated with upregulation of innate and acquired antifungal Th1 responses, such as a dramatically higher production of IL-12, nitric oxide (NO) and TNF-alpha as well as IFN-gamma by CD4+ T cells. Pharmacological inhibition of NO production greatly reduced resistance to gastrointestinal candidiasis, thus pointing to the importance of IL-10-dependent NO regulation at mucosal sites in fungal infections. These results are reminiscent of those obtained in genetically susceptible mice, in which IL-10 administration increased, and IL-10 neutralization decreased, susceptibility to C. albicans and A. fumigatus infections. Collectively, these observations indicate that the absence of IL-10 augments innate and acquired antifungal immunity by upregulating type 1 cytokine responses. The resulting protective Th1 responses lead to a prompt reduction of fungal growth, thus preventing tissue destruction and lethal levels of proinflammatory cytokines.

Interleukin-4 causes susceptibility to invasive pulmonary aspergillosis through suppression of protective type I responses.

Aspergillus fumigatus, an opportunistic fungal pathogen, causes multiple allergic and nonallergic airway diseases. Invasive pulmonary aspergillosis (IPA) is a nonallergic, life-threatening disease of immunocompromised patients. In a murine model of IPA, interleukin (IL)-4-deficient (IL-4-/-) BALB/c mice were used to examine the role of IL-4 in lung pathology and immune responses. IL-4-/- mice were more resistant than wild-type mice to infection caused by multiple intranasal injections of viable A. fumigatus conidia. Resistance was associated with decreased lung inflammatory pathology, impaired T helper (Th)-2 responses (including lung eosinophilia), and an IL-12-dependent Th1 response. In contrast, development of host-detrimental antifungal Th2 cells occurred in IL-12-/- and interferon-gamma-/- mice and in IL-4-/- mice when subjected to IL-12 neutralization. These results demonstrate that IL-4 renders mice susceptible to infection with A. fumigatus by inhibition of protective Th1 responses. IL-4 appears to have a distinct role in the pathogenesis of allergic and nonallergic lung diseases caused by the fungus.

IL-10 is required for development of protective Th1 responses in IL-12-deficient mice upon Candida albicans infection.

IL-12 is both required and prognostic for Th1 development in mice with Candida albicans infection. To delineate further the physiologic role of IL-12 in antifungal immunity, mice deficient for this cytokine were assessed for susceptibility to C. albicans infections, and for parameters of innate and adaptive immunity. IL-12-deficient mice were highly susceptible to gastrointestinal infection or to reinfection and showed elevated production of Candida-specific IgE and IL-4 and defective production of IFN-gamma. The failure to mount protective Th1 responses occurred despite the presence of an unimpaired innate antifungal immune response, which correlated with unaltered IFN-gamma production, but defective production of, and responsiveness to, inhibitory IL-10. IL-10 or IL-12 neutralization increased the innate antifungal resistance in wild-type mice. However, in IL-12-deficient mice, treatment with exogenous IL-12 or IL-10 impaired IL-4 production and increased resistance to infection, through a negative effect on the CTLA-4/B7-2 costimulatory pathway. These results confirm the obligatory role of IL-12 in the induction of anticandidal Th1 responses, and indicate the existence of a positive regulatory loop between IL-12 and IL-10 that may adversely affect the innate antifungal response, but is required for optimal costimulation of IL-12-dependent CD4+ Th1 cells.

Cytokine- and T helper-dependent lung mucosal immunity in mice with invasive pulmonary aspergillosis.

The role of cytokine- and T helper (Th)-dependent lung mucosal antifungal immunity in murine invasive pulmonary aspergillosis (IPA) was investigated. Intact or leukopenic DBA/2 mice were resistant or highly susceptible, respectively, to infection caused by multiple intranasal injections of viable Aspergillus fumigatus conidia. Resistance was associated with unimpaired innate antifungal activity of pulmonary phagocytic cells, concomitant with high-level production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-12 and the presence of interstitial lymphocytes producing interferon-gamma and IL-2. Conversely, production of TNF-alpha and IL-12 was down-regulated in highly susceptible mice, which also had defective innate antifungal immunity and high-level production of IL-4 and IL-10 by lung lymphocytes. Resistance was increased in susceptible mice upon local IL-4 or IL-10 neutralization or IL-12 administration. These results indicate that, similar to observations in mice with disseminated aspergillosis, innate and Th1-dependent immunity play an essential role in host defense against IPA.

IFN-gamma is required for IL-12 responsiveness in mice with Candida albicans infection.

To elucidate the role of IFN-gamma in antifungal CD4+ Th-dependent immunity, 129/Sv/Ev mice deficient for IFN-gamma receptor (IFN-gammaR(-/-)) were assessed for susceptibility to gastrointestinal or systemic Candida albicans infection and for parameters of innate and adaptive T helper immunity. IFN-gammaR(-/-) mice failed to mount protective Th1-mediated acquired immunity upon mucosal immunization or in response to a live vaccine strain of the yeast. The impaired Th1-mediated resistance correlated with defective IL-12 responsiveness, but not IL-12 production, and occurred in the presence of an increased innate antifungal resistance. The development of nonprotective Th2 responses was observed in IFN-gammaR(-/-) mice upon mucosal infection and subsequent reinfection. However, under experimental conditions of Th2 cell activation, the occurrence of Th2 cell responses was similar in IFN-gammaR(-/-) and in IFN-gammaR(+/+) mice. These results indicate the complex immunoregulatory role of IFN-gamma in the induction of mucosal and nonmucosal anticandidal Th cell responses; IFN-gamma is not essential for the occurrence of Th2 responses but is required for development of IL-12-dependent protective Th1-dependent immunity.

Defective co-stimulation and impaired Th1 development in tumor necrosis factor/lymphotoxin-alpha double-deficient mice infected with Candida albicans.

To define the immunological functions of tumor necrosis factor (TNF) in Candida albicans infection, TNF/lymphotoxin (LT)-alpha double-deficient mice were assessed for susceptibility to systemic or gastrointestinal infection and parameters of innate and adaptive Th immunity. When compared to wild-type mice, TNF/LT-alpha-deficient mice were more susceptible to either type of infection caused by virulent or low-virulence C. albicans cells. Susceptibility to infection correlated with impaired development of protective Th1 responses, in spite of the production of bioactive IL-12. The occurrence of predominant Th2 responses was associated with both impaired antifungal effector functions of neutrophils and a defective expression of co-stimulatory molecules on macrophages. All functions were improved upon administration of recombinant TNF-alpha, also resulting in increased resistance to infection. These findings indicate that the protective effect of TNF-alpha in candidiasis relies on the induction of antifungal Th1 responses, possibly occurring through stimulation of antifungal effector functions and co-stimulatory activities of phagocytic cells.

Endogenous interleukin 4 is required for development of protective CD4+ T helper type 1 cell responses to Candida albicans.

Interleukin (IL)-4-deficient mice were used to assess susceptibility to systemic or gastrointestinal Candida albicans infections, as well as parameters of innate and elicited T helper immunity. In the early stage of systemic infection with virulent C. albicans, an unopposed interferon (IFN)-gamma response renders IL-4-deficient mice more resistant than wild-type mice to infection. Yet, IL-4-deficient mice failed to efficiently control infection in the late stage and succumbed to it. Defective IFN-gamma and IL-12 production, but not IL-12 responsiveness, was observed in IL-4-deficient mice that failed to mount protective T helper type 1 cell (Th1)-mediated acquired immunity in response to a live vaccine strain of the yeast or upon mucosal immunization in vivo. In vitro, IL-4 primed neutrophils for cytokine release, including IL-12. However, late treatment with exogenous IL-4, while improving the outcome of infection, potentiated CD4(+) Th1 responses even in the absence of neutrophils. These findings indicate that endogenous IL-4 is required for the induction of CD4(+) Th1 protective antifungal responses, possibly through the combined activity on cells of the innate and adaptive immune systems.

Type 1 and type 2 cytokines: from basic science to fungal infections.

At the present time, it is clear that Th1 responses afford protection against the fungi; however, the development, maintenance and function of the protective immune responses are complex mechanisms and are influenced by multiple factors. The route of infection has been shown to affect initial cytokine production and, consequently, the induction of protective Th1 responses. The ability of different isolates of the same fungal agent to induce and sustain a protective response has also been emphasized. Protective immune responses have been shown to vary in genetically different mouse strains after infection. In addition, these protective responses, such as cellular influx and cytokine production, also vary within the same animal depending on the tissue infected. The functional dominance of certain cytokines over others in influencing development and maintenance of protective responses has been discussed. Certain cytokines may act differently in hosts lacking important components of their innate or immune repertoire. It is evident from these presentations that a more comprehensive understanding of the protective mechanisms against different fungal agents is emerging. However, there is still much to learn before cytokine modulatory therapy can be used effectively without risk in the human host.

Cytokines and mycoses.

Cell-mediated immunity (CMI) has been shown, over many decades of clinical observation and bench research, to be central to the outcome of invasive fungal infections. In recent years, understanding the role of messenger molecules (cytokines), in coordinating and augmenting cellular immunity has been ascendant. These studies have made it possible to consider using cytokines, now available in abundant quantities via recombinant DNA technologies, to treat fungal infections. In this symposium, the most important fungal pathogens that cause infections in humans, particularly in immunocompromised patients, are considered, with emphasis on how recent experimental work may lead to a better understanding of the role of cytokines and their use in therapy.

Induction of protective Th1 responses to Candida albicans by antifungal therapy alone or in combination with an interleukin-4 antagonist.

Resistance or susceptibility to disseminated and mucosal Candida albicans infections in mice correlates with the development of protective or nonprotective T helper (Th) cell responses. To determine whether immunomodulatory activity on Th cell functions is an effect beyond that provided by antifungal therapy, mice with disseminated or gastrointestinal infection were treated with amphotericin B or fluconazole and assessed for mortality, fungus burden in the organs, and parameters of Th cell-dependent immunity. Both antimycotics produced protective CD4+ Th1 cell responses, as revealed by increased production of interleukin (IL)-12 and interferon-y, decreased production of IL-4, delayed-type hypersensitivity to fungal antigen, and the disappearance of antigen-specific IgE. Concomitant neutralization of endogenous IL-4 greatly increased the antifungal efficacy and the Th1-promoting activity of both agents. These results indicate that successful antifungal therapy alone or in combination with cytokine antagonists may rely on the induction of an appropriate Th antifungal cell response.

Neutrophil production of IL-12 and IL-10 in candidiasis and efficacy of IL-12 therapy in neutropenic mice.

Neutrophil defects predispose to severe fungal infections, yet the immunomodulatory role of these cells is poorly defined. The contribution of neutrophils to the early cytokine balance governing Th1 and Th2 cell development was examined in mice with candidiasis. Neutrophils secreted IL-12 and IL-10, correlating with the respective development of self-limiting (Th1-associated) and progressive (Th2-associated) disease. Exogenous IL-12 was effective in protecting neutropenic hosts susceptible to infection. These results suggest that 1) neutrophils, via their ability to release cytokines, play an active role in determining the qualitative development of the T cell response, and 2) their early role in anticandidal immunity can be replaced by exogenous IL-12.

Early T cell unresponsiveness in mice with candidiasis and reversal by IL-2: effect on T helper cell development.

To investigate the role and effect of IL-2 in the genesis of Th1 and Th2 responses to Candida albicans in vivo, we assessed the levels of IL-2 production and the Ag-specific proliferative response in mice with healing or nonhealing infection and the effects of IL-2 neutralization or administration on the course and outcome of infection and on the type of CD4+ Th immunity elicited. High levels of IL-2 production and Ag-specific proliferation in vitro correlated with disease progression in susceptible mice. In contrast, resolution of infection in resistant mice was accompanied by the induction of Ag-specific hyporesponsiveness and impaired IL-2 production. Progression of infection did not occur in susceptible mice treated with anti-IL-2 or anti-IL-2R mAbs; conversely, disease resolution was prevented in resistant mice treated with IL-2. CD4+ Th1 cell responses were present in BALB/c mice rendered resistant by IL-2 neutralization and CD4+ Th2 responses in mice rendered susceptible by IL-2 treatment. The presence of IL-2 restored Ag-specific responsiveness in vitro and correlated in vivo with the expansion of CD4+ MEL-149(low) cells capable of producing IL-2 and IL-4 both in vitro and in vivo as observed in adult thymectomized mice. These results indicate that production of IL-2 early in infection correlates with the induction of IL-4-producing CD4+ Th2 cells, while a transient loss of T cell responsiveness, such as IL-2 production, appears to be required for CD4+ Th1 occurrence in mice with candidiasis.

An immunoregulatory role for neutrophils in CD4+ T helper subset selection in mice with candidiasis.

Granulocytes may serve immunoregulatory and effector roles in different limbs of the immune response to infection. Using live vaccine strain or virulent challenge in mucosal or systemic infection of mice with Candida albicans, we examined the effect of mAb-mediated depletion of neutrophils on the course of primary and secondary challenge and on development of CD4+ cell-dependent immunity. We obtained evidence of deleterious effects of neutrophil depletion occurring at the time of infection under all conditions of testing, both in naive and in previously immunized mice. In contrast, neutrophil depletion appeared to benefit the hosts late in the course of an overwhelming systemic infection. In an attempt to correlate neutrophil function with the nature of the T cell response, we tested the ability of neutrophils to produce cytokines associated with functionally distinct CD4+ Th cell responses to Candida. We found that neutrophils were endowed with the capacity to secrete IL-12 and IL-10 in vitro in response to the yeast. Neutrophil ablation early in the course of Th1-associated, self-limiting infection appeared to change the qualitative development of the T cell response, and rendered mice susceptible to infection. In addition to long recognized contributions to acute anti-candidal responses, these data suggest an important role for neutrophils both in initiation and in expression of Candida-specific immunity.

CD4+ T-helper-cell responses in mice with low-level Candida albicans infection.

Resistance and susceptibility to Candida albicans infection have been shown to be dependent upon the activation of CD4+ T helper (Th) type 1 or Th2 cells, respectively. To study the type, kinetics, and cytokine dependency of CD4+ Th-cell responses in low-level C. albicans infection, susceptible mice were infected with sublethal doses of C. albicans and assessed for parameters of CD4+ Th-dependent immunity. Interleukin (IL)-12 and gamma interferon were always produced early in infection regardless of the pathogen load. In contrast, production of IL-4, and hence Th2-cell reactivity, was strictly dose dependent, being induced at the higher dose of the fungus. Production of IL-12 correlated with a successful control of infection in mice exposed to the lower doses of C. albicans but not with the development of acquired immunity. An antigenic stimulus appeared to be required for IL-12 to induce a protective anticandidal response. Cytokine depletion in vivo revealed that neutralization of IL-4 was protective early but not late in infection, suggesting a different role for IL-4 in the induction versus maintenance of an ongoing anticandidal Th response. Late in infection, an exacerbative effect was also observed upon IL-12 neutralization. These results indicate that the fungal burden and timing of cytokine appearance greatly influence CD4+ Th induction and effector functions in mice with candidiasis.