Trim72 is a major host factor protecting against lethal Candida albicans infection

Abstract

Candida albicans is the most common aetiologic pathogen of fungal infections associated with high mortality in immunocompromised patients. There is an urgent need to develop new antifungal therapies owing to the poor efficacy and resistance of current antifungals. Here, we report that Trim72 positively regulates antifungal immunity during lethal fungal infection. Trim72 levels are significantly increased after Candida albicans infection. In vivo, Trim72 knockout significantly increases mortality, organ fungal burden and kidney damage in mice after lethal Candida albicans infection. Whereas recombinant Trim72 protein treatment protects mice against invasive candidiasis. Mechanistically, Trim72 facilitates macrophage infiltration and CCL2 production, which mediates Trim72-elicited protection against lethal Candida albicans infection. Furthermore, Trim72 may enhance macrophage migration and CCL2 production via NF-κB and ERK1/2 signaling. Inhibition of NF-κB and ERK1/2 signaling abrogates Trim72-mediated protection against lethal Candida albicans infection. Therefore, these data imply that Trim72 may be developed as a host-directed therapy for treating severe systemic candidiasis.

Fig 1. Trim72 expression is upregulated during Candida albicans (C. albicans) infection.

(A) Trim72 levels were measured by ELISA in mice serum and tissue homogenates at the indicated times after C. albicans infection (n = 5 per group). (B) Western blot analysis of Trim72 protein expression in tissue homogenates at the indicated times after C. albicans infection (n = 3 per group). The intensity of the proteins was measured. Data are representative of triplicate independent experiments. Statistical significance was calculated by kruskal-wallis test or one-way ANOVA followed by Dunnett’s or Dunnett T3 multiple comparison test (A, B). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 2. Trim72^-/- mice were susceptible to systemic C. albicans infection.

(A) WT or Trim72^-/- mice were injected intravenously with 3×10⁵ CFU of C. albicans. Survival was recorded (n = 20 per group). (B) The disease score of WT and Trim72^-/- mice was recorded at the indicated times after infection (n = 10 per group). (C) Typical pictures of the condition of WT and Trim72^-/- mice were shown at 2 days after infection. (D) Gross picture of the kidney from WT and Trim72^-/- mice at 2 days after infection (n = 5 per group). (E) Blood urea nitrogen and serum creatinine levels in WT and Trim72^-/- mice at 2 days after infection (n = 5 per group). (F) NGAL mRNA expression in kidney tissue from WT and Trim72^-/- mice at 2 days after infection (n = 5 per group). (G) C. albicans fungal load in organs at 2 days after infection (n = 5 per group). (H) Kidney sections were stained with hematoxylin and eosin (H&E), periodic-acid-Schiff (PAS), or sliver stain at 2 days after C. albicans infection. Representative pictures were presented. Scale bar = 20 um. Data are representative of triplicate independent experiments. Statistical significance was calculated by Log-rank test (A), two-way ANOVA (B), two-tailed unpaired t-test (E-G) or nonparametric Mann Whitney U test (G). Data are presented as mean (B) or mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3. Trim72 treatment protects mice against C. albicans infection.

(A) The study protocol was shown. Mice were injected intraperitoneally with various doses of recombinant murine Trim72 (rmTrim72) (62.5, 125, and 250 μg/kg) or vehicle control 30 min prior to intravenous infection with 3×10⁵ CFU C. albicans, and with 31, 62.5, and 125 μg/kg rmTrim72 respectively at 2 days after infection. (B) Survival of vehicle-treated and various doses of rmTrim72-treated mice (n = 20 per group). (C and D) Weight loss (n = 10 per group) (C) and disease score (n = 5 per group) (D) of vehicle-treated and rmTrim72 (375μg/kg)-treated mice at the indicated times after infection. (E) Typical pictures of the condition of vehicle-treated and rmTrim72 (375μg/kg)-treated mice at 7 days after infection. (F) Gross picture of the kidney from vehicle-treated and rmTrim72 (375μg/kg)-treated mice at 7 days after infection (n = 5 per group). (G) Blood urea nitrogen and serum creatinine levels in vehicle-treated and rmTrim72 (375μg/kg)-treated mice at 7 days after infection (n = 5 per group). (H) NGAL mRNA expression in kidney tissue from vehicle-treated and rmTrim72 (375μg/kg)-treated mice at 7 days after infection (n = 5 per group). (I) C. albicans fungal load in kidney from vehicle-treated and rmTrim72 (375μg/kg)-treated mice at indicated times after infection (n = 5 per group). (J) Kidney sections were stained with H&E, PAS or sliver stain at 7 days after infection. Representative images were shown. Scale bar = 20 um. All data are representative of triplicate independent experiments. Statistical significance was calculated by Log-rank test (B), two-way ANOVA (C and D), two-tailed unpaired t-test (G-I). Data are presented as mean (C and D) or mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4. Trim72 promotes macrophage recruitment and CCL2 production in the infected kidney.

(A) Flow cytometry analysis of CD11b⁺F4/80⁺ macrophages and CD11b⁺Ly6G⁺ neutrophils in the kidneys of WT or Trim72^-/- mice at 2 days after C. albicans infection (n = 5 per group). (B) CCL2 levels in renal tissue homogenates from WT or Trim72^-/- mice detected by ELISA at 2 days after C. albicans infection (n = 5 per group). (C) Flow cytometry analysis of CD11b⁺F4/80⁺ macrophages and CD11b⁺Ly6G⁺ neutrophils in the kidneys of vehicle-treated or rmTrim72 (375μg/kg)-treated mice at 2 and 4 days after C. albicans infection (n = 5 per group). (D) CCL2 levels in renal tissue homogenates from vehicle-treated or rmTrim72 (375μg/kg)-treated mice detected by ELISA at 2 and 4 days after C. albicans infection (n = 5 per group). Data are representative of triplicate independent experiments. Statistical significance was calculated by two-tailed unpaired t-test (A-D). Data are presented as mean ± SD. *p < 0.05, **p < 0.01.

Fig 5. Macrophage-mediated Trim72 provides protection against systemic C. albicans infection in mice.

(A) The study protocol for the administration of rmTrim72 (375μg/kg) and 200ul of clodronate-containing liposomes (CLD) or empty liposomes (Lipo) was shown. Mice were intravenous infected with 3×10⁵ CFU of C. albicans. (B) Survival of vehicle-treated or rmTrim72 (375μg/kg)-treated mice in the presence or absence of macrophages depletion after C. albicans infection (n = 12 per group). (C and D) C. albicans fungal load (C) and CCL2 levels (D) in the kidneys from mice in (A) at 2 days after infection (n = 5 per group). Survival data were collected from three independent experiments. Other data are representative of triplicate independent experiments. Statistical significance was calculated by Log-rank test (B), or one-way ANOVA followed by Dunnett’s multiple comparison test (C, D). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 6. CCL2–macrophage axis mediates Trim72-elicitated protection against systemic C. albicans infection in mice.

(A) Flow cytometry analysis of CD11b⁺F4/80⁺ macrophages in the kidneys of vehicle-treated or Bindarit (100mg/kg)-treated mice at 4 days after C. albicans infection (n = 5 per group). (B) The study protocol for the administration of rmTrim72 (375μg/kg) and Bindarit (100mg/kg) was shown. (C) Survival of mice in (B) (n = 12 per group). (D) C. albicans fungal load in the kidneys from mice in (B) at 4 days after infection (n = 5 per group). (E) Blood urea nitrogen and serum creatinine concentrations in mice in (B) at 4 days after infection (n = 5 per group). (F) CD11b⁺F4/80⁺ macrophages in the kidneys of mice in (B) at 4 days after infection by flow cytometry analysis (n = 5 per group). (G) CCL2 levels in renal tissue homogenates of mice in (B) detected by ELISA at 4 days after infection (n = 5 per group). Survival data were collected from three independent experiments. Other data are representative of triplicate independent experiments. Statistical significance was calculated by two-tailed unpaired t-test (A), Log-rank test (C), or one-way ANOVA followed by Dunnett’s multiple comparison test (D-G). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 7. Trim72 enhances macrophage migration and CCL2 production in vitro.

(A) Rates of phagocytosis for live C. albicans (MOI = 1) by peritoneal macrophages pretreated with vehicle or rmTrim72 (1μg/ml) overnight (n = 5 per group). (B) Phagocytosis for FITC-labeled heat-killed C. albicans (MOI = 1) by peritoneal macrophages pretreated with vehicle or rmTrim72 (1μg/ml) overnight (n = 5 per group). Representative images were shown. Scale bar = 25 um. (C) Rates of killing for live C. albicans (MOI = 1) by peritoneal macrophages pretreated with vehicle or rmTrim72 (1μg/ml) overnight (n = 5 per group). (D) Schematic diagram of the transwell migration assay was shown. Migration of primary peritoneal macrophages treated with vehicle or rmTrim72 (1μg/ml) in a transwell migration assay (n = 5 per group). Representative images were presented. Scale bar = 1000 um. (E) CCL2 levels detected by ELISA in primary peritoneal macrophages treated with vehicle or rmTrim72 (1μg/ml) (n = 5 per group). (F) Rates of phagocytosis for live C. albicans (MOI = 1) by WT or Trim72^-/- peritoneal macrophages (n = 5 per group). (G) Phagocytosis for FITC-labeled heat-killed C. albicans (MOI = 1) by WT or Trim72^-/- peritoneal macrophages. Representative images were shown. Scale bar = 25 um. (H) Rates of killing for live C. albicans (MOI = 1) by WT or Trim72^-/- peritoneal macrophages (n = 5 per group). (I) Migration of primary peritoneal macrophages isolated from WT or Trim72^-/- mice in a transwell migration assay (n = 5 per group). Representative images were shown. Scale bar = 1000 um. (J) CCL2 levels detected by ELISA in primary peritoneal macrophages isolated from WT or Trim72^-/- mice (n = 5 per group). Data are representative of triplicate independent experiments. Statistical significance was calculated by two-tailed unpaired t-test (A-D, F-J) or nonparametric Mann Whitney U test (E). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 8. NF-kB and ERK1/2 signaling pathways regulate macrophage migration and CCL2 production induced by Trim72.

(A and B) Primary peritoneal macrophages pretreated with rmTrim72 (1μg/ml) or vehicle were stimulated with C. albicans for the indicated periods and analyzed by Western blotting for the indicated signaling molecules. Representative images were shown (A). The intensity of the proteins was measured (B) (n = 3 per group). (C and D) WT or Trim72^-/- macrophage were stimulated with C. albicans for the indicated periods and analyzed by Western blotting for the indicated signaling molecules. Representative images were shown (C). The intensity of the proteins was measured (D) (n = 3 per group). (E) Peritoneal macrophages were stimulated with NF-κB inhibitor PDTC (25uM) and ERK1/2 inhibitor U0126 (20uM) or vehicle, followed by rmTrim72 (1μg/ml) or vehicle treatment. Migration of primary peritoneal macrophages was assessed in a transwell migration assay (n = 5 per group). Representative images were shown. Scale bar = 1000μm. (F) CCL2 levels of macrophages in (E) detected by ELISA (n = 5 per group). Data are representative of triplicate independent experiments. Statistical significance was calculated by two-tailed unpaired t-test (B, D), one-way ANOVA followed by Dunnett T3 multiple comparison test (E, F). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 9. Inhibiting NF-κB and ERK1/2 signaling pathways abolished Trim72-mediated protection against invasive C. albicans infection.

(A) The study protocol for the administration of rmTrim72 (375μg/kg), PDTC (100mg/kg) and U0126 (30mg/kg) was shown. Mice were intravenous infected with 3×10⁵ CFU of C. albicans. (B) Survival of mice in (A) (n = 12 per group). (C) C. albicans fungal load in kidneys from mice in (A) at 4 days after infection (n = 5 per group). (D) Blood urea nitrogen and serum creatinine levels in mice in (A) at 4 days after infection (n = 5 per group). (E) CD11b⁺F4/80⁺ macrophages in the kidneys of mice in (A) at 4 days after C. albicans infection by flow cytometry analysis (n = 5 per group). (F) CCL2 levels in renal tissue homogenates from mice in (A) detected by ELISA at 4 days after C. albicans infection (n = 5 per group). Survival data were collected from three independent experiments. Other data are representative of triplicate independent experiments. Statistical significance was calculated by Log-rank test (B), or one-way ANOVA followed by Dunnett’s multiple comparison test (C-F). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 10. Trim72 augments migration capacity and CCL2 production through NF-κB and ERK1/2 signaling in human macrophages.

(A) Trim72 levels were measured by ELISA in serum of patients with candidemia (n = 37) and healthy subjects (n = 20). (B) Trim72 levels in serum of candidemia survivors (n = 25) and candidemia non-survivors (n = 12). (C and D) Rates of phagocytosis (C) and killing (D) for live C. albicans (MOI = 1) by human monocyte-derived macrophages (hMDMs) treated with vehicle or rmTrim72 (1μg/ml) overnight (n = 5 per group). (E) Migration of hMDMs treated with vehicle or rmTrim72 (1μg/ml) in a transwell migration assay (n = 5 per group). (F) CCL2 levels detected by ELISA in hMDMs treated with vehicle or rmTrim72 (1μg/ml) overnight (n = 5 per group). (G) hMDMs were stimulated with NF-κB inhibitor PDTC (25uM) and ERK1/2 inhibitor U0126 (20uM) or vehicle, followed by rmTrim72 (1μg/ml) or vehicle treatment. Migration of hMDMs in a transwell migration assay (n = 5 per group). (H) CCL2 levels of macrophages with the indicated treatments detected by ELISA (n = 5 per group). (I) Schematic representation of the potential mechanism by which Trim72 protects against C. albicans infection. Except for clinical research (A, B), data are representative of triplicate independent experiments. Statistical significance was calculated by nonparametric Mann Whitney U test (A, B), two-tailed unpaired t-test (C-F), or one-way ANOVA followed by Dunnett’s multiple comparison test (I, H). Data are presented as mean (A, B) or mean ± SD (C-H). *p < 0.05, **p < 0.01, *** p < 0.001.