Characteristics of invasive candidiasis in gamma interferon- and interleukin-4-deficient mice: role of macrophages in host defense against Candida albicans

Abstract

Murine models of invasive candidiasis were used to study the in vivo importance of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) in host defense against Candida albicans and to characterize the tissue inflammatory reactions, with special reference to macrophages (Mphi). Knockout (KO) IFN-gamma-deficient (GKO) and IL-4-deficient (IL-4 KO) and C57BL/6 parental mouse strains were challenged intraperitoneally with 10(8) C. albicans blastoconidia. Survival of GKO mice was significantly lower (16.7%) than that of C57BL/6 control (55.5%) and IL-4 KO (61.1%) animals, but was not correlated with the extent of organ colonization. Immunohistological analysis with a panel of myeloid and lymphoid markers revealed multiple renal abscesses, myocarditis, hepatitis, meningoencephalitis, and pneumonia in each strain, with a dominant presence of Mphi. In the absence of IFN-gamma, C. albicans induced striking changes in the phenotype of alveolar Mphi and extensive perivascular lymphoid infiltrates in the lung. Impairment in nitric oxide production by peritoneal Mphi was shown only in GKO mice, and they produced Candida-specific immunoglobulin G (IgG), IgM, IgA, and IgG subclasses in lower titers. Our in vivo studies with KO mice elucidate a critical role for IFN-gamma, but not IL-4, in host defense against C. albicans.

FIG. 1

Survival of GKO, IL-4 KO, and C57BL/6 control mice challenged i.p. with 10⁸ C. albicans blastoconidia. Survival of GKO mice was significantly lower than that of IL-4 KO and C57BL/6 control animals from day 14 (P < 0.05 at days 21 and 28 for both). Each group contained 40 animals initially. The figure represents pooled data of two separate sets of experiments, which gave results in close agreement.

FIG. 2

CFU of C. albicans from various organs of GKO, IL-4 KO, and C57BL/6 control mice after i.p. challenge with 10⁸ Candida blastoconidia. Each bar represents the mean ± standard error. Experiments were performed in duplicate with five or more mice.

FIG. 3

Histological appearance of lesions in the kidneys (A to D) and heart (E and F) at day 3 of murine invasive candidiasis. Inflammatory lesions in both the kidneys and heart consisted of mostly FA/11⁺ macrophages (A, C, and E). Yeast and mycelial forms of fungus were shown in the lesions by Gomori’s silver methenamine staining and were both extra- and intracellular (B, D, and F). The histopathologies of the kidneys and heart in GKO, IL-4 KO, and C57BL/6 control mice were similar. Representative photographs were taken of sections from IL-4 KO (A and B) and GKO mice (C to F). Bar, 50 μm.

FIG. 4

Histopathology of the liver in invasive candidiasis at day 14. Perivascular infiltrates contained MHC II⁺ cells (A) and included CD3⁺ (B), CD4⁺ (D), and CD8⁺ (F) T cells and FA/11⁺ Mφ (C); only a few B220⁺ B cells (E) were associated with the lesions. The histopathologies of the liver in GKO, IL-4 KO, and C57BL/6 control mice were similar; representative photographs of liver in C57BL/6 mice are shown. Bar, 50 μm.

FIG. 5

Histopathology of the lungs in invasive murine candidiasis at day 3 in GKO (A to H) and C57BL/6 mice (I to L). Inflammatory lesions in GKO mice at day 2 (M and N) and day 14 (O and P) are also shown. Perivascular infiltrates appeared only in GKO mice and contained MHC II⁺ (TIB120⁺) mononuclear cells (B), CD3⁺ T cells (C), mostly CD4⁺ (D) and fewer CD8⁺ (E) cells and B220⁺ B cells (F), which were also CD19⁺ (not shown). Mφ FA/11⁺ (G) were not a dominant component of the infiltrates, but were present in two different phenotypes in the lungs: elongated, perivascular cells and engorged, diffusely distributed alveolar Mφ (H). Perivascular infiltrates contained only debris of C. albicans, as shown by Gomori’s staining (A). Histopathology of the lungs in IL-4 KO and C57BL/6 mice was markedly different from that of GKO mice; a similar inflammatory reaction did not develop in these strains even at later stages of infection. Lung sections obtained at day 3 from C57BL/6 mice were stained with TIB120 (I), FA/11 (J), B220 (K), and anti-CD3 (L) MAbs and showed only minimal inflammatory responses. At day 2, perivascular MHC II⁺ cell aggregates (M) appeared in GKO mice, most of which were CD3⁺ T cells (N), and by day 14, the extension of perivascular infiltrates was reduced compared with that of day 3, containing MHC II⁺ cells (O), which were partly CD3⁺ T cells (P). Bars, 50 μm.

FIG. 6

Nitrite concentration in 48-h culture supernatants of peritoneal Mφ isolated from C57BL/6, IL-4 KO and GKO infected mice at day 5 of infection. The nitrite production of peritoneal macrophages from IL-4 KO mice was significantly higher than that of the control. The impaired function of Mφ from GKO animals could be restored by ex vivo IFN-γ treatment for 48 h (100 U/ml). The height of each bar represents the mean ± standard error of three experiments performed in duplicate.

FIG. 7

(A) C. albicans-specific IgM, IgG, and IgA titers in sera of mice infected i.p. with 10⁸ Candida blastoconidia. Pooled sera of at least four mice/group were analyzed for specific antibodies by standard ELISA. The height of each bar represents the mean ± standard error of three experiments performed in duplicate. (B) C. albicans-specific IgG1, IgG3, IgG2a, and IgG2b titers in sera of mice infected i.p. with 10⁸ Candida blastoconidia. Pooled serum samples of at least four mice per group were analyzed for specific antibodies by standard ELISA. The height of each bar represents the mean ± standard error of three experiments performed in duplicate.