Comparison of pathogenesis and host immune responses to Candida glabrata and Candida albicans in systemically infected immunocompetent mice

Abstract

Cytokine-mediated host defense against Candida glabrata infection was compared to that against C. albicans, using immunocompetent murine models of systemic candidiasis. The pathogenesis of infection was evaluated morphologically and by culture of target organs, while the kinetics of induction of cytokine mRNAs and corresponding proteins were determined in kidneys by real-time reverse transcription-PCR and cytokine-specific murine enzyme-linked immunosorbent assays, respectively. Systemic infection with C. glabrata resulted in a chronic, nonfatal infection with recovery of organisms from kidneys, while intravenous inoculation with C. albicans resulted in rapid mortality with logarithmic growth of organisms in kidneys and recovery of C. albicans from the spleen, liver, and lungs. Survival of C. glabrata-infected mice was associated with rapid induction of mRNAs and corresponding immunoreactive proteins for the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-12 (IL-12), and gamma interferon (IFN-gamma) and the lack of induction of protein for the anti-inflammatory cytokine IL-10. In contrast, mortality in C. albicans-infected mice was associated with induction of mRNA and corresponding protein for IL-10 but delayed (i.e., TNF-alpha) or absent (i.e., IL-12 and IFN-gamma) induction of immunoreactive proinflammatory cytokines. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to TNF-alpha, IL-12, or IFN-gamma, and the effect on growth of C. glabrata in kidneys was assessed. Neutralization of endogenous TNF-alpha resulted in a significant increase in C. glabrata organisms compared to similarly infected mice administered an isotype-matched control MAb, while neutralization of endogenous IL-12 or IFN-gamma had no significant effect on C. glabrata replication. These results demonstrate that in response to intravenous inoculation of C. glabrata, immunocompetent mice develop chronic nonfatal renal infections which are associated with rapid induction of the proinflammatory cytokines TNF-alpha, IL-12, and IFN-gamma. Furthermore, TNF-alpha plays a key role in host defense against systemic candidiasis caused by either C. glabrata or C. albicans, as the absence of endogenous TNF-alpha activity was associated with enhanced tissue burden in both infection models.

FIG. 1

Growth of C. glabrata and C. albicans in tissues of immunocompetent Crl:CF-1 mice. Mice were inoculated i.v. with C. glabrata (10⁸ organisms/mouse) or with C. albicans (5 × 10⁶ organisms/mouse). At specific time points p.i., mice were euthanized, and net growth of C. glabrata and C. albicans was quantified in kidneys, hearts, lungs, brains, spleens, livers, and lungs by culture of tissue homogenates. Results represent the mean ± SEM of CFU (log₁₀)/tissue of three mice per time point.

FIG. 2

Pathology in Crl:CF-1 mice systemically inoculated with C. glabrata or C. albicans. (A) Mouse kidney 4 h after infection with C. glabrata. Note organisms (arrows) in macrophages attached to the renal vessel endothelium. (B) Mouse kidney 72 h after infection with C. glabrata. Note organisms (arrow) in renal glomeruli with a mixed mononuclear cell infiltrate. (C) Mouse kidney 24 h after infection with C. albicans. Note organisms (arrow) in glomeruli with moderate mononuclear cell infiltrate. (D) Mouse heart 24 h after infection with C. albicans. Note organisms (arrow) and polymorphonuclear infiltrate. (E) Mouse brain 48 h after infection with C. albicans. Note organisms (arrow) with minimal inflammatory response. (F) Mouse spleen 48 h after infection with C. albicans. Note organisms (arrow) with mild mononuclear cell infiltrate. Magnification, ×370.

FIG. 3

Temporal expression of TNF-α, IL-12, IFN-γ, and IL-10 mRNAs in kidneys of C. glabrata- or C. albicans-infected mice. Crl:CF-1 mice were infected i.v. with virulent C. glabrata (10⁸ CFU/mouse) or C. albicans (5 × 10⁶ CFU/mouse). At specific time points p.i., mice were euthanized, kidneys were excised, and total RNA was extracted. Transcript levels for TNF-α (a), IL-12 p40 (b), IL-12 p35 (c), IFN-γ (d), and IL-10 (e) were quantified in kidneys by real-time RT-PCR. For mRNA quantification, PCR amplification of the housekeeping ubiquitin gene was performed for each sample to control for sample loading and facilitate normalization between samples. The ubiquitin-normalized data were expressed as fold induction of gene expression in C. glabrata- or C. albicans-infected mice compared to uninfected mice. Results represent the mean ± SEM of two separate experiments, four to nine mice per treatment group. ∗, significantly greater than control mice, P < 0.05.

FIG. 4

Temporal expression of TNF-α, IL-12, IFN-γ, and IL-10 proteins in kidneys during C. glabrata and C. albicans infection. Crl:CF-1 mice were infected with virulent C. glabrata or C. albicans as described for Fig. 3. At specific time points p.i., the mice were euthanized, and kidneys were excised and homogenized. Levels of the immunoreactive cytokines TNF-α (a), IL-12 p70 (b), IFN-γ (c), and IL-10 (d) were quantified in kidney homogenates. Results represent the mean ± SEM of two separate experiments, 8 to 14 mice per treatment group. ∗, significantly greater than control mice, P < 0.05.

FIG. 5

Role of endogenous TNF-α, IL-12, and IFN-γ in resolution of primary systemic C. glabrata infection. Crl:CF-1 mice were administered control (IgGa) (), TNF-α (), anti-IL-12 (), or anti-IFN-γ () MAb (1 mg/mouse intraperitoneally) 1 day prior to infection with C. glabrata. At 3, 5, and 7 days p.i., mice were euthanized, kidneys were excised and homogenized, and growth of C. glabrata was quantified by culture of kidney homogenates. Results represent the pooled mean ± SEM of two separate experiments, 10 to 15 animals per treatment group. *, significantly greater than similarly infected mice administered control MAb, P < 0.05.