HECT E3 Ubiquitin Ligase Nedd4 Is Required for Antifungal Innate Immunity

Abstract

Candida albicans is the most common cause of fungal infections in humans, and disseminated candidiasis has become one of the leading causes of hospital-acquired bloodstream infections with a high mortality rate. However, little is known about the host-pathogen interactions and the mechanisms of antifungal immunity. Here, we report that Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) is essential for signaling through Dectin-1 and Dectin-2/3. We showed that mice that lack Nedd4 globally or only in the myeloid compartment are highly susceptible to systemic C. albicans infection, which correlates with heightened organ fungal burden, defective inflammatory response, impaired leukocyte recruitment to the kidneys, and defective reactive oxygen species expression by granulocytes. At the molecular level, Nedd4 ^-/- macrophages displayed impaired activation of TGF-β-activating kinase-1 and NF-κB, but normal activation of spleen tyrosine kinase and protein kinase C-δ on C. albicans yeast and hyphal infections. These data suggest that Nedd4 regulates signaling events downstream of protein kinase C-δ but upstream of or at TGF-β-activating kinase-1.

Figure 1:. Construction of the targeting vector and generation of the Floxed nedd4 allele in mice:

(a) schematic structure of the mouse nedd4 wild type allele and the targeting vector. The top line represent the genomic DNA structure containing exons 1-5 of the mouse wild type nedd4 allele and the lower line represents the recombinant allele. The targeting vector was constructed for conditional deletion of exons 2 and 3 in the plasmid containing both LoxP and Frt cassettes; (b) Western blots of tissue homogenates from various organs of Nedd4^f/fROSA^CreER mice pretreated with corn oil or tamoxifen and harvested 3 days later; (c) Western blots of tissue homogenates from various organs from Nedd4^f/fROSA^CreER mice pretreated with oil (0) or tamoxifen which were harvested 1, 2, or 3 weeks after the last injection; (d) Western blots of lysate from immune cell subsets T (T cells), B (B cells), DC (DC cells) and M (macrophages) from Nedd4^f/f (WT), CD11c^Cre.Nedd4^f/f (dcKO), and LysM^Cre.Nedd4^f/f (mKO) mice. Data are representative of 2 independent experiments.

Figure 2:. Nedd4 regulates CLRs but not TLRs signaling:

(a) Control Nedd4^f/f and Nedd4^ΔM BMDMs were stimulated with TLR ligands 1 to 9, α-mannan, zymosan, and curdlan for 24 h; TNFα (left) and IL-6 (right) levels in the supernatant were detected using ELISA. Data are pooled from 3 independent experiments with three replicates of each sample per experiment. Error bars are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired two-tailed Student’s t test. (b-c) BMDMs (top) or BMNs (bottom) from control Nedd4^f/f and Nedd4^ΔM were infected with C. albicans hyphae (b) or yeast (c) at MOI of 1:1 for 0, 1 or 3 hrs; (d) control Nedd4^f/f and Nedd4^ΔM BMDM were infected with Conidia from A. fumigatus. TNF-α and IL-6 content in supernatant was measured using ELISA. For Fig 2B – 2D, data are pooled from 3 independent experiments with three replicates of each sample per experiment. Error bars are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired two-tailed Student’s t test.

Figure 3:. Nedd4 deficiency impairs ROS production and fungal killing in BMDMs and BMNs:

(a) ROS production by control Nedd4^f/fROSA^CreER-Oil or Nedd4^f/fROSA^CreER-Tam BMDM (left) and BMNs (right) was determined by co-culture with C. albicans yeast at MOI = 5:1 and monitored in real time over the indicated time period using chemiluminescence assay. Values are expressed as relative light units (RLU) and represent mean values of 4 replicates in each sample. Data were analyzed using student’s t-test; (b) control Nedd4^f/f or Nedd4^ΔM BMDM were co-cultured with C. albicans yeast (cap1 mutant) at an MOI of 40 :1 for 24 hrs (left), control Nedd4^f/f or Nedd4^ΔM BMN were co-cultured with C. albicans yeast (cap1 mutant) at an MOI of 5 :1 for 2 hrs (right). The fungal/BMDM or fungal/BMN homogenates were plated on YPD media overnight and fungal killing determined. Data are pooled from 3 independent experiments, with each group having 4 samples per experiment. Error bars are mean ± s.d. **P < 0.01, ***P < 0.001 by Student’s t test; (c) Control Nedd4^f/f or Nedd4^ΔM BMDMs were co-cultured with ConA Alexa Fluor 488–labeled C. albicans. After 45 min of co-culture, control Nedd4^f/f or Nedd4^ΔM BMDMs were analyzed for C. albicans uptake by flow cytometry. Data are pooled from 2 independent experiments, with each group having 4 samples per experiment. Error bars are mean ± s.d.

Figure 4:. Nedd4 deficient mice have reduced survival and higher fungal burden during C. albicans infection:

(a) Mice lacking Nedd4 in myeloid cells (left) or systemically (right) and control Nedd4^f/f were injected with 5 x 10⁵ CFU of C. albicans through the tail vein and monitored for survival; Kaplan-Meier survival curve was plotted to determine survival rate. Data are are pooled from three independent experiments with 5 mice per group per experiment. P< 0.05 by Log-rank test. (b-c) Control Nedd4^f/f and Nedd4^ΔM mice were injected with 2.5 x 10⁵ CFU of C. albicans through the tail vein for 48 hrs, (b) serum TNFα and IL-6 production at the stated times were determined by ELISA. Data are are pooled from three independent experiments with 5 mice per group per experiment. Error bars are mean ± s.d. **P < 0.01 ***P < 0.001; by unpaired two-tailed Student’s t-test. (c) Another set of mice were sacrificed 48 hrs post infection and the kidney, spleen, liver and lung homogenates were plated on YPD media for 24 hrs then colonies were counted and fungal burden determined. Data are are pooled from three independent experiments with 5 mice per group per experiment. Error bars are mean ± s.d. **P < 0.01 ***P < 0.001; by unpaired two-tailed Student’s t-test (d) Kidney histopathology analysis by H&E staining (top) and PAS staining (bottom) to visualize fungal burden (hyphae); (n = 5 mice per group). Images are representative of two independent experiments (biological replicates). Scale bars, 100 μm.

Figure 5:. Nedd4 enhances effector functions of innate immune cells:

Control Nedd4^f/f and Nedd4 deficient mice were injected with 2.5 x 10⁵ CFU of C. albicans through the tail vein for 48 hrs. (a) Immune cell recruitment to the kidney was measure by flow cytometry; (b) ROS production by myeloid cells in the kidney and spleen measured using flow cytometry. For Fig 5A and 5B, data are pooled from three independent experiments. For each experiment there were 5 mice per group, each with three replicate samples. Error bars are mean ± s.d. *P < 0.05, unpaired two-tailed Student’s t test.

Figure 6:. Nedd4 is required for TAK1 and NF-κB but not Syk and PKC-δ activation:

(a-b) Immunoblot analysis of the indicated proteins from the cell lysate of control Nedd4^f/f and Nedd4^ΔM BMDMs after infection with hyphae (a) and yeast (b) from C. albicans. Images are representative of three independent experiments (biological replicates).