Inhibiting the immunoproteasome exacerbates the pathogenesis of systemic Candida albicans infection in mice

Abstract

Apart from its role in MHC class I antigen processing, the immunoproteasome has recently been implicated in the modulation of T helper cell differentiation under polarizing conditions in vitro and in the pathogenesis of autoimmune diseases in vivo. In this study, we investigated the influence of LMP7 on T helper cell differentiation in response to the fungus Candida albicans. We observed a strong effect of ONX 0914, an LMP7-selective inhibitor of the immunoproteasome, on IFN-γ and IL-17A production by murine splenocytes and human peripheral blood mononuclear cells (PBMCs) stimulated with C. albicans in vitro. Using a murine model of systemic candidiasis, we could confirm reduced generation of IFN-γ- and IL-17A-producing cells in ONX 0914 treated mice in vivo. Interestingly, ONX 0914 treatment resulted in increased susceptibility to systemic candidiasis, which manifested at very early stages of infection. Mice treated with ONX 0914 showed markedly increased kidney and brain fungal burden which resulted in enhanced neutrophil recruitment and immunopathology. Together, these results strongly suggest a role of the immunoproteasome in promoting proinflammatory T helper cells in response to C. albicans but also in affecting the innate antifungal immunity in a T helper cell-independent manner.

Figure 1. Influence of ONX 0914 on C. albicans-induced production of IL-6, IL-17A, and IFN-γ by murine splenocytes and human PBMCs in vitro.

Culture supernatant levels of IL-6, IL-17A, and IFN-γ were measured by ELISA. (A) Naive murine splenocytes were pulsed with DMSO or 200 nM ONX 0914 for 2 h and cultured in the presence of heat-killed C. albicans yeast for up to 6 days. Data are representative for one out of three independent experiments and expressed as mean +/− SD. (B) Human PBMCs from healthy donors were treated as described in (A) and cultured in the presence of heat-killed C. albicans hyphae for 5 days. Data represent blood samples from three different donors. Data are analyzed by two-way ANOVA with *p < 0.05 and **p < 0.01.

Figure 2. LMP7 inhibition reduces the development of IL-17A and IFN-γ producing cells in vivo.

Mice were i.v. infected with 1 × 10⁵ CFU live C. albicans blastoconidia and treated with 10 mg/kg ONX 0914 (s.c.) every second day. On day 7 postinfection, splenocytes were restimulated with 10⁶/ml heat-killed C. albicans yeast cells for 48 h. Levels of IL-17A (left graph) and IFN-γ (right graph) were measured in the supernatant by ELISA. Data are representative for one out of three independent experiments and expressed as mean +/− SEM of n = 5 mice (n = 2 naive mice). Data are analyzed by two-way ANOVA with *p < 0.05 and ***p < 0.001.

Figure 3. Influence of the immunoproteasome on weight loss and survival in systemic candidiasis.

C57BL/6, LMP7^−/−, LMP2^−/−, and MECL-1^−/− mice were i.v. infected with 1 × 10⁵ CFU live C. albicans blastoconidia and treated with vehicle or 10 mg/kg ONX 0914 (s.c.) every second day where indicated (B,C). (A,C) Change of body weight during systemic infection with C. albicans. Percent weight loss (y-axis) is plotted versus time (x-axis). Data points represent mean weight change +/− SEM. (B) Survival curves of ONX 0914 and vehicle treated mice. Data represent combined results from 2–3 experiments with n = 8–18 mice per group. Data are analyzed by two-way ANOVA with *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4. Influence of LMP7 inhibition on fungal burden in kidney, liver, and brain.

Mice were intravenously infected with 1 × 10⁵ CFU live C. albicans blastoconidia and treated with vehicle or 10 mg/kg ONX 0914 (s.c.) every second day. On day 3 (left panels) and day 7 (right panels) fungal burden was determined in kidneys (A), brains (B), and livers (C). Bars show mean log₁₀ CFU/g tissue +/− SEM. Data represent pooled results from two independent experiments and are analyzed by non-parametric Mann-Whitney test with *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 5. Elevated neutrophil numbers in the kidney and the brain of ONX 0914 treated mice.

Mice were i.v. infected with 1.5 × 10⁵ CFU live C. albicans blastoconidia and treated with vehicle or 10 mg/kg ONX 0914 (s.c.) every second day. Kidneys and brains were removed and isolated leukocytes were stained for Ly6-G, CD45, F4/80, CD11b, MHC-II, and CD11c and analyzed by flow cytometry (gated on living cells according to FSC/SSC). Graphs show (A) representative flow cytometry profiles of kidney infiltrating CD45^intLy6-G^high neutrophils on day 7, (B) pooled results from two independent experiments presented as mean percentage of kidney infiltrating CD45^intLy6-G^high neutrophils +/− SEM, (C,D) pooled results from two independent experiments presented as mean absolute numbers +/− SEM of (C) kidney infiltrating CD45^intLy6-G^high neutrophils, MHCII^-F4/80^intCD11b⁺ monocytes, MHCII⁺ F4/80⁺ CD11b⁺ macrophages, and CD11b⁺MHCII^highCD11c⁺ myeloid derived dendritic cells (mDCs) 48 h p.i., and (D) brain infiltrating CD45^intLy6-G^high neutrophils, CD45^highCD11b⁺ myeloid cells, CD11b⁺MHCII^highCD11c⁺ mDCs, and CD45^intCD11b⁺ CNS resident microglia 72 h p.i. Data were analyzed by students t test with *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6. Influence of ONX 0914 treatment on neutrophil recruitment.

Mice were i.v. infected with 1 × 10⁵ CFU live C. albicans blastoconidia and treated with vehicle or 10 mg/kg ONX 0914 (s.c.) every second day. (A) Peripheral blood (day 3, 5, and 7) and (B) bone marrow cells were stained for Ly6-G and CD45 and analyzed by flow cytometry. Graphs show mean percentage of CD45^intLy6-G^high neutrophils (gated on living cells according to FSC/SSC) +/− SEM. (C) Serum levels of KC (CXCL1) were determined by cytometric bead array. Graphs show pooled data from two independent experiments and are presented as mean +/− SEM. (D,E) Relative mRNA expression of KC (CXCL1), MIP-1α (CCL3), MCP-1 (CCL2), or MIP-2α (CCXL2) in the kidney on day 2 (D) or day 7 p.i. (E). Real-time RT-PCR data are pooled from two independent experiments with n = 8–10 mice per group and expressed as fold induction over naive +/− SEM relative to one out of four naive mice, which served as uninfected control. Data were analyzed by students t test with **p < 0.01.

Figure 7. Influence of LMP7 inhibition on the maintenance of kidney function and proinflammatory serum cytokine levels.

Mice were i.v. infected with 1 × 10⁵ CFU live C. albicans blastoconidia and treated with vehicle or 10 mg/kg ONX 0914 (s.c.) every second day. Graphs show (A) photometrically determined serum levels of urea and creatinine on day 7 and (B) serum levels of IL-6 and TNF-α as detected by cytometric bead array on day 3. (C) Relative mRNA expression of KIM-1 on day 2 and day 7, pooled from two independent experiments with n = 8–10 mice per group, and presented as fold induction over naive +/− SEM relative to one out of four naive mice, which served as uninfected control. Data are analyzed by students t test with *p < 0.05.

Figure 8. Influence of LMP7 inhibition on C. albicans-induced activation of innate immune cells.

(A) Mice were intravenously infected with 1.5 × 10⁵ CFU live C. albicans-GFP blastoconidia and treated with vehicle or 10 mg/kg ONX 0914 (s.c.) on the day of infection. 48 h p.i., kidneys were removed and isolated leukocytes were stained for Ly6-G and CD45. Graph shows mean percentages of GFP⁺CD45^intLy6-G^high cells or median fluorescence of GFP in CD45^intLy6-G^high cells +/− SEM, respectively. (B–E) Mice were i.v. infected with 1.5 × 10⁵ CFU live C. albicans blastoconidia and treated with vehicle or 10 mg/kg ONX 0914 (s.c.) on the day of infection. Graphs show median fluorescence +/− SEM of CD11b expression on (B) kidney or (D) brain infiltrating CD45^intLy6-G^high neutrophils and MHC class II expression on (C) kidney infiltrating MHC-II⁺F4/80⁺CD11b⁺ macrophages or (E) brain infiltrating CD45^high CD11b⁺ myeloid cells, respectively. Data were pooled from two independent experiments (n = 7–10 mice per group). Data were analyzed by students t test with *p < 0.05 and ***p < 0.001. (F) NADPH oxidase activity as determined by DHR test. Isolated human neutrophils were treated with DMSO or 200 nM ONX 0914 in vitro for one hour and stimulated with 140 nM PMA for 30 min. Graph shows result from one experiment representative for three different blood donors.