IFN-γ Is Required for IL-12 Responsiveness in Mice with Candida albicans Infection

Elio Cenci, Antonella Mencacci, Giuseppe Del Sero, Cristiana Fé d’Ostiani, Paolo Mosci, Angela Bacci, Claudia Montagnoli, Manfred Kopf and Luigina Romani
J Immunol October 1, 1998, 161 (7) 3543-3550;

Abstract

To elucidate the role of IFN-γ in antifungal CD4+ Th-dependent immunity, 129/Sv/Ev mice deficient for IFN-γ receptor (IFN-γR−/−) were assessed for susceptibility to gastrointestinal or systemic Candida albicans infection and for parameters of innate and adaptive T helper immunity. IFN-γR−/− 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-γR−/− 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-γR−/− and in IFN-γR+/+ mice. These results indicate the complex immunoregulatory role of IFN-γ in the induction of mucosal and nonmucosal anticandidal Th cell responses; IFN-γ is not essential for the occurrence of Th2 responses but is required for development of IL-12-dependent protective Th1-dependent immunity.

Helper T cells play a central role in regulating immune responses to the fungus Candida albicans by providing critical cytokine-mediated activation and deactivation signals to fungicidal effector cells (1, 2, 3). In experimental models of candidiasis, protection correlates with the generation of CD4+ Th1 cells producing IL-2 and IFN-γ (4) and requires the concerted action of several cytokines (5, 6, 7, 8), including IL-12 (9, 10, 11). Susceptibility to infection correlates with the generation of CD4+ Th2 cells producing IL-4 and IL-10 (4, 12, 13, 14) that have been implicated in inhibiting the development of protective Th1 cells (15) and in opposing the IFN-γ-mediated activation of fungicidal macrophages (16).

Early studies in vitro indicating IFN-γ as a major activating factor for fungicidal phagocytes (17) have been confirmed by recent in vivo data (18). Analysis of cytokine gene expression in PBMC from healthy humans stimulated in vitro with Candida Ags has revealed appreciable levels of IFN-γ mRNA (19). Overproduction of IFN-γ may also be involved in the acute pathology of fungal septic shock (11). Besides its effector function, the role of IFN-γ in candidiasis may encompass early regulatory effects on Th cell differentiation, leading to the onset of protective immunity (5, 20). IFN-γ is rapidly produced after infection in both resistant and susceptible mice (9). Ag-specific secretion of IFN-γ protein in vitro by CD4+ cells occurs only in genetically resistant mice (10), such that its neutralization prevents the development of protective Th1 responses (5). However, the mechanisms underlying the Th1-promoting activity of IFN-γ in candidiasis are not fully understood. High endogenous levels of IFN-γ are not sufficient per se to inhibit anticandidal Th2 responses (10), thus suggesting a minimal down-regulatory effect on Th2 differentiation. IFN-γ might have been required for initial production of IL-12. However, production of IL-12 by phagocytic cells in response to Candida occurs in the absence of IFN-γ even though it is potentiated by priming with IFN-γ (11).

IFN-γ exerts multiple and complex effects on the regulation of Th induction and effector functions. It was found to be either required (21, 22) or not required (23, 24) for IL-12-dependent Th1 differentiation of CD4+ T cells and to exert antiproliferative effects on Th2 cells (25) due to their ability to express the β-chain of the IFN-γR (26). The lack of this expression would make Th1 cells resistant to IFN-γ by preventing transduction of its signal (26, 27). IFN-γ could act also as an activator or a death signal for T lymphocytes (28, 29), thus regulating both Th1 and Th2 responses (30). Recently, a novel and important role has been ascribed to IFN-γ: its ability to maintain IL-12 responsiveness in Th1 cells by overriding IL-4-induced inhibition of IL-12β2 receptor expression (31).

The availability of the inbred 129/Sv/Ev strain of mice lacking a functional IFN-γ system (32) by inactivation of the α-chain receptor (IFN-γR−/−)3 allows us to elucidate the role of IFN-γ in CD4+ Th cell development in experimental candidiasis. IFN-γR−/− and wild-type mice were infected under conditions that otherwise result in the induction of either protective Th1 or nonprotective Th2 antifungal immune responses. Mice were assessed for susceptibility to infection as well as for parameters of innate and mucosal and nonmucosal Th immunity. IFN-γ exerted complex effects on the regulation of immunity to C. albicans, affecting both innate and acquired Th1-dependent immune responses.

Materials and Methods

Mice

The mutant (129/Sv/Ev) mouse strain deficient in IFN-γR α-chain (IFN-γR−/−) or IL-4 (IL-4−/−) were generated by gene targeting in murine embryonic stem cells as previously described (32, 33, 34). IFN-γR−/− mice develop a normal immune response, possess IFN-γ-independent macrophage and NK cell activities, and constitutively express MHC Ag. However, they lack a functional IFN-γ system (33). In these experiments we used IFN-γR−/− and wild-type 129/Sv/Ev mice of both sexes (bred under specific pathogen-free conditions at the breeding facilities of the University of Perugia, Perugia, Italy). For each experiment, groups of mice were matched for sex and age as much as possible. Procedures involving animals and their care were conducted in conformity with national and international laws and policies.

Yeasts, infections, in vivo analysis, and treatments

The origin and characteristics of the C. albicans highly virulent strain and the low virulence live vaccine strain used in this study have been described in detail previously (4, 6). For infection, cells were washed twice in saline and diluted to the desired density to be injected i.v. via the lateral tail vein in a volume of 0.5 ml/mouse or intragastrically (108 cells) via an 18-gauge 4-cm-long plastic catheter as previously described (35, 36). The viability of the cells was >95% by trypan blue dye exclusion test and quantitative cultures. Resistance to reinfection was assessed by injecting mice with 106 virulent Candida cells i.v. 14 days after primary infection. Quantification of yeast cells in organs of infected mice (six to eight per group) was performed by a plate dilution method, using Sabouraud dextrose agar, and results (mean ± SEM) were expressed as CFU per organ.

For histology, tissues were excised and immediately fixed in formalin. Sections (3–4 mm) of paraffin-embedded tissues were stained with periodic acid-Schiff reagent and examined for histology as previously described (35, 36). Mice succumbing to yeast challenge were routinely necropsied for histopathologic confirmation of disseminated candidiasis. Soluble IL-4R (Behringwerke, Marburg/Lahn, Germany) was given i.p. at 80 μg/injection on days 1, 3, and 5 after infection as previously described (13).

Purification and culture of cells

CD4+ T splenocytes were positively selected by sequential adherence on anti-Ig-coated plates and then on anti-murine CD4 (mAb GK1.5); splenic macrophages (referred to as adherent or accessory macrophages) were obtained by 2-h plastic adherence as previously described (4, 6). CD4+ T cells were cultured (5 × 106) in the presence of 5 × 105 accessory macrophages and 5 × 105 heat-inactivated yeast cells. Intragastric lymphocytes (IGL) were isolated from the whole stomach after incubation in calcium and magnesium ion-free HBSS containing DTT (0.145 μg/ml; Sigma, St. Louis, MO) and EDTA (0.37 μg/ml) and further digestion in RPMI 1640 containing collagenase VIII (100 U/ml; Sigma) and DNase I (0.1 μg/ml, Sigma) as described previously (8). Lymphocyte-enriched populations, isolated at the 40/100% interface of a discontinuous Percoll gradient (Pharmacia, Uppsala, Sweden) were used in a spot ELISA (see below) to enumerate cytokine-producing cells. Cultures of splenic adherent macrophages and purified peritoneal neutrophils, collected 18 h after i.p. inoculation of aged, endotoxin-free 10% thioglycolate solution (Difco, Detroit, MI), were performed as previously described (37, 38) by incubating 5 × 105 cells in the presence of 400 U/ml IFN-γ and 40 ng/ml LPS (Sigma). Cytokine measurement was performed in supernatants collected after 48 h (for lymphocytes and macrophages) and 24 h (for neutrophils) as previously described (37, 38).

Cytokine, Ab, and ELISPOT assays

The levels of IFN-γ, IL-2, and IL-4 in culture supernatants were determined by means of cytokine-specific ELISAs, using pairs of anti-cytokine mAbs, as previously described (9, 10). The Ab pairs used were as follows, listed by capture/biotinylated detection: IFN-γ, R4–6A2/XMG1.2; IL-2, JES6-1A12/JES6-5H4; and IL-4, BVD4-1D11/BVD6-24G2.3 (PharMingen, San Diego CA). For IL-12 p70 measurement, a modified Ab capture bioassay was used (7). Cytokine titers were calculated by reference to standard curves constructed with known amounts of recombinant cytokines (from PharMingen or Genetics Institute, Cambridge, MA, for IL-12). A micro-ELISA procedure was used to quantitate yeast-specific IgG2a, IgG1, and IgE in the sera of mice as previously described (4). The procedure used to enumerate cytokine-producing cells was based on an ELISPOT assay, as previously described (7), using pairs of anti-cytokine mAbs as described above, including the TRFK-5/TRFK-4 pair of mAbs for IL-5, and freshly isolated IGL cultured without further stimulation.

Candidacidal assay

For the candidacidal assay, 5 × 105 splenic adherent macrophages or elicited peritoneal neutrophils were incubated with 105 C. albicans cells for 4 or 1 h, respectively, and the number of CFU was determined as previously described (16). The percentage of CFU inhibition (mean ± SE) was determined as the percentage of colony formation inhibition = 100 − (CFU experimental group/CFU control cultures) × 100.

RT-PCR

RNA extraction and amplification of synthesized cDNA from macrophages and purified CD4+ splenocytes were performed as previously described (8, 9). For hypoxanthine-guanine phosphoribosyl transferase (HPRT), IL-12p40, IL-12Rβ1, and IL-12Rβ2, the primers, positive controls, cycles, and temperature were previously described (8, 9). The HPRT primers were used as a control for both RT and the PCR reaction itself and also for comparing the amount of products from samples obtained with the same primer. The PCR fragments were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. PCR-assisted mRNA amplification was repeated at least twice for at least two separately prepared cDNA samples for each experiment. Data are representative of three different experiments.

Statistical analysis

Survival data from each group of wild-type mice were compared with those from mutant mice using the Mann-Whitney U test; significance was defined as p < 0.05. Student’s t test was used to determine statistical significance between the two groups for cytokine production and organ clearance. In vivo groups consisted of four to six animals. The data reported are from three experiments.

Results

Susceptibility of IFN-γR−/− mice to gastrointestinal C. albicans infection and systemic reinfection

To assess the functional role of IFN-γ in an infectious condition that otherwise results in the development of protective Th1 responses (35, 36), IFN-γR−/− and IFN-γR+/+ mice were injected intragastrically with 108 virulent C. albicans cells and monitored for resistance to mucosal infection and systemic reinfection in terms of survival and fungal growth in the organs (Table I⇓). Although survival to gastrointestinal infection did not differ in either type of mice, IFN-γR−/− mice were more susceptible to infection; local fungal growth in the stomach was significantly higher than that observed in wild-type mice at 4 wk after infection. Histopathologic examination of stomach sections from IFN-γR−/− mice (Fig. 1⇓A) revealed the presence of numerous intraepithelial abscesses throughout the nonglandular portion of the stomach starting from the cardial-atrial fold region, which is a major site of C. albicans colonization in the murine gastrointestinal tract (35, 36). Abscesses consisted of a thickened keratinized outer layer that enveloped a dense aggregate of hyphae and infiltrating inflammatory cells. Conspicuous signs of acanthosis and hypercheratosis were also visible. In wild-type mice (Fig. 1⇓B), fewer yeast cells were present in the keratinized layer, with a few intraepithelial abscesses, a limited inflammatory reaction at the mucosal level, and no signs of extensive acanthosis and hypercheratosis. On assaying resistance to systemic reinfection, wild-type, but not IFN-γR−/−, mice showed increased resistance, as evidenced by survival and decreased fungal burden in the kidneys (Table I⇓). Histopathologic examination of the kidneys of IFN-γR−/− mice (Fig. 1⇓C) revealed extensive fungal growth associated with the presence of numerous foci of inflammatory reaction (mainly consisting of fungal and polymorphonuclear cells) throughout the kidney parenchyma, as opposed to the reduced fungal growth and few healing lesions, consisting mainly of mononuclear cells, observed in the kidneys of IFN-γR+/+ mice (Fig. 1⇓D). These data indicate that in the absence of IFN-γ signaling, mice are highly susceptible to mucosal C. albicans infection and are incapable of developing resistance to systemic reinfection.

FIGURE 1.
FIGURE 1.

Pathologic analysis of organ tissues of IFN-γR−/− and IFN-γR+/+ mice with candidiasis. Periodic acid-Schiff-stained sagittal sections from the stomach of IFN-γR−/− (A) or IFN-γR+/+ (B) mice at 4 wk after intragastric infection with C. albicans. In IFN-γR−/− mice (A), note the presence of numerous intraepithelial abscesses (indicated by arrows) containing fungal elements and inflammatory cells (magnified in the inset) throughout the nonglandular region of the stomach. Conspicuous signs of acanthosis and hypercheratosis were also visible. In wild-type mice (B), fewer yeast cells were present in the keratinized layer, with a few intraepithelial abscesses (arrows), a limited inflammatory reaction, and no signs of extensive acanthosis and hypercheratosis. C and D, Sections of the kidneys of intragastrically infected IFN-γR−/− (C) and IFN-γR+/+ (D) mice 3 days after systemic reinfection. Numerous abscesses (arrows), consisting of fungal and polymorphonuclear cells (magnified in the inset), were visible throughout the kidney parenchyma of IFN-γR−/− mice (C) as opposed to few healing lesions (indicated by arrows and magnified in the inset) observed in wild-type mice (D). Bars: A through D, 400 μm; inset, 50 μm. Boxes indicate the areas magnified in insets.

View this table:
Table I.

C. albicans gastrointestinal infection and reinfection in IFN-γR+/+ and IFN-γR−/− mice

Susceptibility of IFN-γR−/− mice to C. albicans infection is associated with failure to mount a protective Th1 response

To assess the type of Th cell response elicited upon gastrointestinal infection and reinfection, mice were monitored for patterns of cytokine and Ab production. Enumeration of cytokine-producing cells in IGL isolated from mice with gastrointestinal infection revealed that the numbers of IL-4- and IL-5-producing cells were decreased in IFN-γR−/− mice compared with wild-type mice. However, the number of IFN-γ-producing cells was also decreased (Fig. 2⇓). The number of IFN-γ- and IL-2-producing cells was decreased in the draining mesenteric lymph nodes (data not shown). Similarly, upon systemic reinfection, CD4+ splenocytes from IFN-γR−/− mice produced less IFN-γ and IL-2 and more IL-4 than those from IFN-γR+/+ mice (Fig. 3⇓A). In addition, serum levels of Ag-specific IgE were higher while those of IgG2a were lower in IFN-γR−/− than wild-type mice. Similar levels of IgG1 were detected (Fig. 3⇓B). These findings were different from those observed in mice upon primary systemic infection, in that production of IFN-γ and particularly IL-4 and levels of IgE were equally elevated in both types of mice (data not shown). These results clearly show the failure of IFN-γR−/− mice to mount protective anticandidal Th1 responses.

FIGURE 2.
FIGURE 2.

Cytokine production in IFN-γR−/− (▪) and IFN-γR+/+ (□) mice, uninfected (none) or intragastrically infected with C. albicans. Cytokine-producing intragastric T lymphocytes were enumerated by ELISPOT assay at 4 wk after infection.

FIGURE 3.
FIGURE 3.

Cytokine and Ab production in IFN-γR−/− (▪) and IFN-γR+/+ (□) mice upon C. albicans reinfection. Mice were intragastrically infected with 108 C. albicans cells and i.v. reinfected 14 days later with 106 yeast cells. A, Three days after reinfection, splenic CD4+ T cells were Ag-stimulated in vitro in the presence of accessory macrophages, and cytokine levels were determined in culture supernatants by means of cytokine-specific ELISA. Cytokine levels in culture supernatants of unstimulated responder cells were below the detection limit of the assay, indicated by < on the y-axis. B, Serum levels of Ag-specific IgE, IgG2a, and IgG1 in mice infected as described in A. The assay was performed 7 days after reinfection. Mice with gastrointestinal infection and no reinfection are denoted None.

IL-12 responsiveness, but not IL-12 production, is impaired in IFN-γR−/− mice with C. albicans infection

The inability of IFN-γR−/− mice to mount Th1 responses upon C. albicans infection could be due to defective IL-12 production, defective IL-12 responsiveness, or both. To distinguish among these possibilities, splenic macrophages and CD4+ T cells from mice with gastrointestinal infection were assessed for expression of IL-12 p40 and IL-12Rβ1 and IL-12Rβ2 chains, respectively, at 3 days after systemic reinfection. The results show that the expression of the IL-12 p40 gene was not impaired in IFN-γR−/− compared with IFN-γR+/+ mice (Fig. 4⇓) nor was the production of bioactive IL-12 by splenocytes impaired upon Ag-specific stimulation in vitro (data not shown). Interestingly, IL-12 p40 gene expression appears to be up-regulated in IFN-γR−/− mice, either uninfected or upon gastrointestinal infection. In contrast, expression of the β2-chain of the IL-12R was down-regulated in IFN-γR−/− mice, with and without reinfection, but not in wild-type mice. The results also show that the constitutive expression of the IL-12Rβ2 mRNA was different in uninfected control mice, being apparently increased in IFN-γR−/− mice. A similar finding was observed in IL-12 p40−/− mice (unpublished observation). No changes were observed in either type of mouse in expression of IL-12Rβ1 (Fig. 4⇓). Both receptor subunits were similarly expressed in the two types of mouse upon primary i.v. infection (data not shown). These data indicate that a defective IL-12 responsiveness, rather than IL-12 production, could account for the inability of mice with impaired IFN-γ signaling to mount anticandidal Th1 responses.

FIGURE 4.
FIGURE 4.

IL-12 p40, IL-12β1, and IL-12β2 receptor gene expression in macrophages (IL-12 p40) and CD4+ T cells (IL-12Rs) from IFN-γR−/− and IFN-γR+/+ mice uninfected (lane 1), with gastrointestinal infection (lane 2), or with gastrointestinal infection and systemic reinfection (lane 3). Macrophages and CD4+ T cells were positively selected from spleens 3 days after reinfection. Cytokine and cytokine receptor gene expression were assessed by RT-PCR. C, HPRT-, cytokine-, or cytokine receptor-specific control. N, no DNA added to the amplification mix during PCR.

IFN-γ is not essential for the development of anticandidal Th2 responses

The observation that failure to activate anticandidal Th1 responses was concomitant with the expression of Th2-dependent immunity in IFN-γR−/− mice prompted us to investigate whether IFN-γ could also exert an inhibitory effect on the development of CD4+ Th2 responses. For this purpose, IFN-γR−/− and IFN-γR+/+ mice were injected i.v. with low virulence C. albicans and were monitored for survival and cytokine production. Wild-type mice were highly susceptible to infection, as indicated by 14-day survival (Table II⇓) and by extensive fungal growth in the kidneys (data not shown). On assaying cytokine production at 7 days after infection, Ag-activated CD4+ splenocytes produced IFN-γ, but relatively low levels of IL-2 and IL-4. However, IL-4 neutralization by treatment with soluble IL-4R significantly increased resistance to infection. Similarly, 129/Sv/Ev IL-4-deficient mice were highly resistant to infection and, importantly, to subsequent reinfection with virulent C. albicans cells. Both IFN-γ and IL-2 productions by CD4+ T cells were increased. These data indicate that the high susceptibility of 129/Sv/Ev to acute C. albicans infection is associated with the occurrence of inhibitory IL-4-producing Th2-cells, such that in the absence of IL-4, development of protective Th1 responses occurred. On infecting IFN-γR−/− mice, no significant changes in cytokine production were observed compared with that in wild-type mice. Analysis of IFN-γ and IL-4 gene expression by quantitative RT-PCR confirmed the results obtained in terms of actual cytokine production, as the levels of IFN-γ and IL-4 gene expression were not different in the two types of mice early in the course of the infection (data not shown). Therefore, the increased resistance to infection does not correlate with decreased Th2 reactivity, as production of IL-4 occurs similarly in both types of mice. We found higher innate resistance to infection in mutant compared with wild-type mice, although they were unable to develop resistance to reinfection. Figure 5⇓A shows that the candidacidal activity of macrophages and neutrophils from uninfected mice was higher in IFN-γR−/− than in IFN-γR+/+ mice, although this activity declined later in the course of infection. We also found that production of IL-12 was not detected in macrophages and was only minimally detected in neutrophils from IFN-γR+/+ mice as opposed to the higher production observed, particularly in neutrophils, in IFN-γR−/− mice (Fig. 5⇓B). These data clearly point to a minimal role, if any, for IFN-γ in inhibiting the development of Th2 responses in mice with C. albicans infection and confirm its requirement for the development of protective Th1 responses.

FIGURE 5.
FIGURE 5.

Effector and immunomodulatory functions of macrophages and neutrophils from IFN-γR−/− (▪) and IFN-γR+/+ (□) mice, uninfected (none) or upon primary i.v. infection with C. albicans. Three days after infection, splenic adherent macrophages and elicited peritoneal neutrophils were assessed for the ability to kill yeast cells (A) and to secrete IL-12 p70 (B). Assays were performed as described in Materials and Methods.

View this table:
Table II.

Course of systemic C. albicans infection and cytokine production in IFN-γR+/+ and IFN-γR−/− mice

Discussion

This study illustrates the complex immunoregulatory role of IFN-γ in the induction of mucosal and nonmucosal anticandidal Th cell responses. While not necessary for the induction and expression of Th2 cell responses, IFN-γ appeared to be an essential requirement for the development of protective anticandidal Th1 cell responses, a finding confirming the result obtained by IFN-γ neutralization in genetically resistant mice (5).

In murine candidiasis, both resistance to mucosal infection and subsequent systemic reinfection are dependent on the activation of Th1 cell responses (4, 35, 36). IFN-γR−/− mice were highly susceptible to gastrointestinal infection and were unable to develop resistance to subsequent reinfection. Susceptibility to mucosal infection correlated with a decreased frequency of intragastric T lymphocytes producing both Th1 (IFN-γ) and Th2 (IL-4 and IL-5) cytokines. Decreased production of IFN-γ and IL-2 was also observed in draining lymph nodes, clearly indicating the failure of IFN-γR−/− mice to mount anticandidal Th1 responses at the mucosal level. In systemic reinfection, susceptibility correlated with a defective production of IFN-γ and IL-2 by CD4+ T splenocytes upon Ag-specific stimulation in vitro.

Failure to mount protective anticandidal Th1 responses was not associated with a decreased production of IL-12, which occurred in these mice after mycobacterial (39) or viral (40) infections. This finding is in line with our previous observation indicating that production of IL-12 by phagocytic cells in response to Candida occurs in the absence of IFN-γ even though it is potentiated by priming with this cytokine (11). In contrast, impaired IL-12 responsiveness was observed, as expression of the IL-12β1 subunit receptor was unaffected but that of IL-12Rβ2 subunit was defective in IFN-γR−/− mice.

That both IL-12β1 and IL-12β2 receptor subunits are essential components of the functional mouse IL-12R (41) and that IFN-γ is needed to override IL-4-induced inhibition of IL-12β2 receptor expression in Th1 cells have recently been reported (31). Our results suggest that in murine candidiasis the Th1-promoting activity of IFN-γ may rely on its ability to maintain IL-12 responsiveness rather than to induce IL-12 production. Although IL-12 is required for Th1 development in mice with candidiasis (9, 10), we have recently shown that IL-12 responsiveness, rather than production, correlates with the occurrence of Th1 cell responses (8).

In the course of the present study we found that the number of CD4+ splenocytes significantly decreased in IFN-γR−/− mice soon after primary systemic infection (data not shown). As IFN-γ has been shown to act as an activator or a death signal for T lymphocytes (28, 29), other mechanisms of IFN-γ-dependent regulation of Th1 induction and effector functions in mice with candidiasis cannot be excluded.

The essential requirement for IFN-γ in Th1 development found in 129/Sv/Ev IFN-γR−/− mice with candidiasis appears to be in contrast with the occurrence of a Th1-type response observed in these mice upon parasitic (42) or viral (43) infection. However, the finding that they are resistant to Th1-mediated experimental autoimmune encephalomyelitis (44) indicates the complex role for IFN-γ in mediating Th1 induction and expression in this particular strain. This observation would be in line with the concept of genetic differences in default pathways for Th1 and Th2 development (45), which may include the diverse influence of IFN-γ on differentiation of T cell subsets in different mouse strains. In this regard, it has been recently hypothesized that IFN-γ may have an obligate role in directing the Th1 phenotype only under conditions where IL-12 production is limiting (46).

Given the antiproliferative effects of IFN-γ on Th2 cells (25), an increased Th2 cell activity in IFN-γR−/− mice upon C. albicans infection was expected. However, numerous studies indicate that the production of IL-4 in the absence of IFN-γ or IFN-γ signaling appears to be dependent on the genetic background and the type of infection. Indeed, administration of IFN-γ mAb during infection with Leishmania major (22) or Listeria monocytogenes (47) leads to disease progression associated with the appearance of Th2 cells. Likewise, infection of mice with a disrupted IFN-γ gene with L. major (48), influenza virus (49), or Mycobacterium tuberculosis (50) increased the production of IL-4 and IL-5. In C57BL6 IFN-γR−/− mice, increased production of IL-4 was observed upon infection by an intracellular pathogen (51). However, studies in 129/Sv/Ev IFN-γR−/− mice failed to demonstrate a default of CD4+ cells to the Th2 phenotype upon viral (43), parasitic (42), or bacterial (52) infection.

We found that production of Th2 cytokines was locally decreased in IFN-γR−/− mice with gastrointestinal infection, thus confirming the occurrence of impaired Th2-dependent mucosal immune response in these mice, as previously reported (53). The susceptibility of IFN-γR−/− mice to systemic reinfection was associated with an up-regulated expression of Th2 cell responses, as revealed by high level production of IL-4 and IgE. However, on directly assessing the functional role of IFN-γ in anticandidal Th2 cell development, we found that IFN-γ is not essential for the development of anticandidal Th2 responses.

Although 129/Sv/Ev mice develop protective Th1 responses to many parasitic, bacterial, and viral infections (52), we found that 129/Sv/Ev mice are highly susceptible to acute infection with low virulence Candida cells and succumb to it. Although IL-4 production was barely detectable upon infection, a common finding observed in other infections (42, 43), resistance to infection was dramatically increased in the absence of IL-4 or upon IL-4 neutralization, clearly showing a pathogenetic role for IL-4-producing Th2 cells in the susceptibility of mice to infection. On assaying the pattern of Th cell responses in similarly infected IFN-γR−/− mice, a comparable level of Th2 cell reactivity was observed, as revealed by IL-4 protein production and cytokine gene expression by quantitative PCR. These results confirm previous observations indicating that high levels of endogenous IFN-γ are not sufficient per se to inhibit anticandidal Th2 responses (10). They also indicate that IFN-γ is not essential for the occurrence of anticandidal Th2 cell responses, a finding in line with that obtained in mice with a disrupted IFN-γ gene and infected with C. albicans under conditions of Th2 cell activation (54).

Despite the occurrence of Th2 cell responses, IFN-γR−/− mice were more resistant to primary acute systemic infection than wild-type mice. A similar finding was observed in IFN-γ-deficient mice (54). This could indicate that mechanisms of innate resistance may efficiently contribute to the early control of infection in IFN-γR−/− mice. However, other studies have indicated an impaired macrophage activation in these mice (32, 55), which was considered to contribute to their susceptibility to certain infections. We found that while the number of circulating neutrophils increased similarly in wild-type and mutant mice after infection (data not shown), the candidacidal activity of macrophages and neutrophils was higher in IFN-γR−/− than in wild-type mice, declining later in the course of infection. In wild-type mice no signs of activation of these cells were observed upon infection. Production of nitric oxide is either increased (55) or decreased (56) in 129/Sv/Ev IFN-γR−/− mice; we were unable to measure production of nitric oxide in either type of mouse upon infection (data not shown). Thus, a prompt and efficient antifungal effector activity of phagocytic cells may account for the increased resistance of IFN-γR−/− mice to the acute infection.

Given the importance of cytokines acting on or secreted by macrophages and neutrophils during infection (57), including C. albicans infection (37, 38), it is likely that a differential production of cytokines with activating or deactivating activity on phagocytic cells may account for the different levels of antifungal effector function observed in mutant and wild-type mice. We found that the production of IL-10 was lower in macrophages and neutrophils from mutant compared with wild-type mice (data not shown). As IL-10 is a potent inhibitor of antifungal effector functions of IFN-γ-activated phagocytic cells (16) and inhibits IL-12 synthesis in these cells (57), the defective production of IL-10 could account for the high level activation of antifungal effector function and IL-12 production in mutant mice.

Preliminary results in wild-type mice treated with IL-10-neutralizing mAb show an increased resistance to the primary systemic infection associated with an increased candidacidal activity of effector phagocytic cells, a finding confirming the suppressive role of IL-10 in C. albicans infection (15). Given that IFN-γ acts as a negative regulator of IL-10 transcription (58), it will be of interest to investigate mechanisms by which deficiencies in IFN-γ signaling lead to decreased IL-10 production in mice with C. albicans infection.

In conclusion, this study shows that IFN-γ is not essential for the occurrence of Th2 responses but is required for development of IL-12-dependent protective Th1 responses in mice with C. albicans infection. Our results confirm those of other experimental models of fungal infection (59) and suggest the importance of IFN-γ signaling at the levels of innate and acquired immune responses to fungi.

Acknowledgments

We thank Eileen Zannetti for excellent secretarial and editorial assistance, Genetics Institute for IL-12, and Dr. Roberto Peducci of the animal facility at the University of Perugia for excellent technical assistance.

Footnotes

  • ↵1 This work was supported by the National Research Program on AIDS–Opportunistic Infections and Tuberculosis, contract 50A.0.28, Italy. The Basel Institute for Immunology was founded and supported by F. Hoffmann-La Roche, Ltd. (Basel, Switzerland).

  • ↵2 Address correspondence and reprint requests to Dr. Luigina Romani, Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy.

  • ↵3 Abbreviations used in this paper: IFN-γR−/−, IFN-γ receptor deficient; IGL, intragastric lymphocytes; ELISPOT, enzyme-linked immunospot; HPRT, hypoxanthine-guanine phosphoribosyl transferase.

  • Received December 27, 1997.
  • Accepted May 27, 1998.

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The Journal of Immunology
Vol. 161, Issue 7
1 Oct 1998
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