Complement C3 plays an essential role in the control of opportunistic fungal infections

Abstract

The innate recognition of fungal pathogens is a crucial first step in the induction of protective antifungal immunity. Complement is thought to be one key component in this process, facilitating fungal recognition and inducing early inflammation. However, the roles of the individual complement components have not been examined extensively. Here we have used mice lacking C3 to examine its role in immunity to opportunistic fungal pathogens and show that this complement component is essential for resistance to infections with Candida albicans and Candida glabrata. We demonstrate that the absence of C3 impairs fungal clearance but does not affect inflammatory responses. We also show that the presence of C3 contributes to mortality in mice challenged with very high doses of Saccharomyces cerevisiae, although these effects were found to be mouse strain dependent.

FIG. 1.

C3 deficiency increases susceptibility to infection with C. albicans. C57BL/6 wild-type (•) and C3-deficient (○) mice were infected with a low (1 × 10⁴ CFU), intermediate (2 × 10⁴ CFU), or high (1 × 10⁵ CFU) dose of C. albicans i.v. and were monitored for survival over 21 days, as described in Materials and Methods. The numbers of mice infected in each experiment are indicated in the figure and are representative of at least two independent experiments (1 × 10⁴ and 2 × 10⁴ CFU) or a single experiment (1 × 10⁵ CFU). *, P < 0.05.

FIG. 2.

C3 deficiency correlates with increased inflammation and fungal burdens. On day 3 following i.v. infection with a low dose (1 × 10⁴ CFU) of C. albicans, higher fungal burdens (A) and exacerbated inflammatory cytokines (B) were observed in the kidneys of C3-deficent mice (○) compared to wild-type (wt) animals (•). The values are as follows: RANTES, 145 ± 16 versus 39 ± 4 pg/ml; G-CSF, 1,054 ± 228 versus 290 ± 120 pg/ml; KC, 326 ± 32 versus 147 ± 57 pg/ml; IL-1β (IL-1b), 2,936 ± 492 versus 1,184 ± 290 pg/ml; macrophage inhibitory protein 1α (MIP-1a), 265 ± 51 versus 57 ± 23 pg/ml; IL-1α (IL-1a), 127 ± 24 versus 44 ± 16 pg/ml; macrophage inhibitory protein 1β (MIP-1b), 27 ± 5 versus 9 ± 2 pg/ml; and IL-17, 3.6 ± 0.6 versus 1.3 ± 0.2 pg/ml. Higher levels of selected proinflammatory cytokines were also detected in the peripheral blood of these animals (C). The values are as follows: G-CSF, 414 ± 176 versus 2 ± 1 pg/ml; KC, 199 ± 34 versus 59 ± 12 pg/ml; IL-1α, 23 ± 2 versus 9 ± 2 pg/ml; and IL-17, 141 ± 9 versus 5 ± 3 pg/ml. Although there was a significant increase in polymorphonuclear leukocyte accumulation in the kidneys of the C3^−/− animals, as quantified by histology (not shown) and measurement of MPO activity (D), there was no difference in the gross pathologies of these tissues, as determined by hematoxylin and eosin staining (E). *, P < 0.05 (n > 7 animals per group per experiment). The data shown in panel A are pooled from two independent experiments. Bars indicate mean values of the data.

FIG. 3.

C3 deficiency does not affect early inflammatory responses to C. albicans. C3^−/− and wild-type mice were infected i.p. with 1 × 10⁶ CFU of C. albicans, and inflammatory responses were measured at 4 h. (A) Incubation of isolated peritoneal fluid (gray histogram) or serum (unfilled histogram) with Candida albicans leads to C3 deposition on the fungal surface. The dark-gray portion of the histogram indicates unstained particles (secondary-only control). (B) Representative flow cytometric profiles showing the gates used (25) to identify recruited neutrophil (NØ) and monocyte/macrophage (Mo) populations. C3 deficiency does not affect cellular recruitment (wild type, 5.71 × 10⁶ ± 4.31 × 10⁵ total cells/ml) (C) or reduce inflammatory cytokine production (D) following i.p. administration of C. albicans. The values for the wild type are as follows: RANTES, 10.5 ± 2.0 pg/ml; G-CSF, 2,218 ± 434 pg/ml; granulocyte-macrophage CSF (GM-CSF), 29.7 ± 3.7 pg/ml; macrophage inhibitory protein 1α (MIP-1a), 313 ± 36 pg/ml; tumor necrosis factor (TNF), 211 ± 37 pg/ml; IL-6, 6,161 ± 1,169 pg/ml; monocyte chemoattractant protein 1 (MCP-1), 5,062 ± 941 pg/ml; IL-1α (IL-1a), 28.8 ± 3.6 pg/ml; IL-10, 43.7 ± 10.0 pg/ml; and macrophage inhibitory protein 1β (MIP-1b), 167 ± 12 pg/ml. Values for the wild type versus C3^−/− are as follows: eotaxin, 3,557 ± 538 versus 7,368 ± 811 pg/ml; KC, 539 ± 142 versus 1,124 ± 180 pg/ml; and IL-1β (IL-1b), 77 ± 14 versus 120 ± 11 pg/ml. (E) C3 deficiency also has no effect on inflammation following i.p. infections with higher doses (1 × 10⁷) CFU of C. albicans (wild type, 6.02 × 10⁶ ± 9.32 × 10⁵ total cells/ml). *, P < 0.05 (n > 6 animals per group per experiment). The data shown in panel C are pooled from three independent experiments. Bars indicate mean values of the data.

FIG. 4.

C3 deficiency does not affect late inflammatory responses to C. albicans. C3^−/− and wild-type mice were infected i.p. with 1 × 10⁶ CFU of C. albicans, and inflammatory responses were measured at day 4. (A) Representative flow cytometric profile showing the gate used (25) to identify recruited macrophages. C3 deficiency does not affect cellular recruitment (B) or inflammatory cytokine production (C) following i.p. administration of C. albicans. Values for the wild type versus C3^−/− are as follows: 1.18 × 10⁷ ± 1.97 × 10⁶ versus 6.65 × 10⁶ ± 1.20 × 10⁶ total cells/ml. Values for the wild type are as follows: RANTES, 5.0 ± 0.4 pg/ml; G-CSF, 80 ± 16 pg/ml; KC, 4.1 ± 1.1 pg/ml; granulocyte-macrophage CSF (GM-CSF), 1.3 ± 0.3 pg/ml; IL-1β (IL-1b), 10.9 ± 3.5 pg/ml; macrophage inhibitory protein 1α (MIP-1a), 29.5 ± 18.0 pg/ml; tumor necrosis factor (TNF), 115 ± 25 pg/ml; IL-6, 2.1 ± 1.1 pg/ml; monocyte chemoattractant protein 1 (MCP-1), 98 ± 46 pg/ml; IL-1α (IL-1a), 15.7 ± 1.8 pg/ml; IL-10, 5.3 ± 1.2 pg/ml; and macrophage inhibitory protein 1β (MIP-1b), 2.8 ± 1.0 pg/ml. *, P < 0.05 (n > 6 animals per group per experiment). The data shown in panel B are pooled from three independent experiments. Bars indicate mean values of the data.

FIG. 5.

C3 deficiency delays fungal clearance. C3^−/− and wild-type mice were infected i.v. with 1 × 10⁴ CFU of C. albicans, and fungal burdens in various organs were determined at 24, 48 and 72 h. *, P < 0.05 (n = 6 animals per group). Bars indicate mean values of the data. Values for 24-h wild type versus C3^−/− are as follows: heart, 234.6 ± 107 versus 212,755 ± 212,165 CFU/g; lung, 262 ± 104 versus 532 ± 175 CFU/g; liver, 137 ± 12 versus 217 ± 68 CFU/g; brain, 217 ± 68 versus 966 ± 367 CFU/g; spleen, 1,505 ± 296 versus 5,834 ± 2,912 CFU/g; kidney, 10,350 ± 2,998 versus 1.86 × 10⁵ ± 6.0 × 10⁴ CFU/g. Values for 48-h wild type versus C3^−/− are as follows: heart, <100 versus 141 ± 28 CFU/g; lung, <100 versus 309 ± 100 CFU/g; liver, 128 ± 23 versus 222 ± 58 CFU/g; brain, 1,007 ± 907 versus 2,260 ± 1,399 CFU/g; spleen, 593 ± 120 versus 10,000 ± 1,576 CFU/g; kidney, 57,823 ± 26,218 versus 3.8 × 10⁵ ± 1.2 × 10⁵ CFU/g. Values for 72-h wild type versus C3^−/− are as follows: heart, <100 versus 181 ± 81 CFU/g; lung, <100 versus 926 ± 515 CFU/g; liver, 112 ± 12 versus 211 ± 61 CFU/g; brain, 154.3 ± 54 versus 572 ± 192 CFU/g; spleen, 459 ± 113 versus 1,672 ± 791 CFU/g; and kidney, 9,833 ± 5,412 versus 2.4 × 10⁵ ± 9.4 ± 10⁴ CFU/g.

FIG. 6.

C3 deficiency increases susceptibility to infection with C. glabrata. C57BL/6 wild-type (•) and C3-deficient (○) mice were infected with 7 × 10⁷ CFU of C. glabrata i.v. and were monitored for survival over 21 days, as described in Materials and Methods. The data shown are pooled from two independent experiments. *, P < 0.05.

FIG. 7.

C3 and C5 deficiency protects against lethality induced upon high-dose infection with S. cerevisiae. C57BL/6 wild-type or C3-deficient, DBA/2, and BALB/c mice were infected with 1 × 10⁸ CFU of S. cerevisiae i.v. and were monitored for survival over 21 days, as described in Materials and Methods. The data shown are pooled from two independent experiments. *, P < 0.05.