Elise M.N. Ferre

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System.

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 -/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 -/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.

Autoimmune Regulator Deficiency Results in a Decrease in STAT1 Levels in Human Monocytes

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare primary immunodeficiency disorder typically caused by biallelic autoimmune regulator (AIRE) mutations that manifests with chronic mucocutaneous candidiasis (CMC) and autoimmunity. Patients with STAT1 gain-of-function (GOF) mutations also develop CMC and autoimmunity; they exhibit increased STAT1 protein levels at baseline and STAT1 phosphorylation (pSTAT1) upon interferon (IFN)-γ stimulation relative to healthy controls. AIRE interacts functionally with a protein that directly regulates STAT1, namely protein inhibitor of activated STAT1, which inhibits STAT1 activation. Given the common clinical features between patients with AIRE and STAT1 GOF mutations, we sought to determine whether APECED patients also exhibit increased levels of STAT1 protein and phosphorylation in CD14+ monocytes. We obtained peripheral blood mononuclear cells from 8 APECED patients and 13 healthy controls and assessed the levels of STAT1 protein and STAT1 tyrosine phosphorylation at rest and following IFN-γ stimulation, as well as the levels of STAT1 mRNA. The mean STAT1 protein levels in CD14+ monocytes exhibited a ~20% significant decrease in APECED patients both at rest and after IFN-γ stimulation relative to that of healthy donors. Similarly, the mean peak value of IFN-γ-induced pSTAT1 level was ~20% significantly lower in APECED patients compared to that in healthy controls. The decrease in STAT1 and peak pSTAT1 in APECED patients was not accompanied by decreased STAT1 mRNA or anti-IFN-γ autoantibodies; instead, it correlated with the presence of autoantibodies to type I IFN and decreased AIRE−/− monocyte surface expression of IFN-γ receptor 2. Our data show that, in contrast to patients with STAT1 GOF mutations, APECED patients show a moderate but consistent and significant decrease in total STAT1 protein levels, associated with lower peak levels of pSTAT1 molecules after IFN-γ stimulation.

VT-1161 protects mice against oropharyngeal candidiasis caused by fluconazole-susceptible and -resistant Candida albicans

Background Candida albicans, the most common human fungal pathogen, causes chronic mucosal infections in patients with inborn errors of IL-17 immunity that rely heavily on chronic, often lifelong, azole antifungal agents for treatment. However, a rise in azole resistance has predicated a need for developing new antifungal drugs. Objectives To test the in vitro and in vivo efficacy of VT-1161 and VT-1129 in the treatment of oropharyngeal candidiasis with azole-susceptible or -resistant C. albicans strains. Methods MICs of VT-1161, VT-1129 and nine licensed antifungal drugs were determined for 31 Candida clinical isolates. The drug concentrations in mouse serum and tongues were measured following oral administration. IL-17-signalling-deficient Act1-/- mice were infected with fluconazole-susceptible or fluconazole-resistant C. albicans strains, and the amount of mucosal fungal burden was determined after fluconazole or VT-1161 treatment. Results Fourteen isolates (45%) were not fluconazole susceptible (MIC ≥4 mg/L). VT-1161 and VT-1129 showed significant in vitro activity against the majority of the 31 mucosal clinical isolates (MIC50 0.03 and 0.06 mg/L, respectively), including Candida glabrata (MIC50, 0.125 and 0.25 mg/L, respectively). After oral doses, VT-1161 and VT-1129 concentrations in mouse serum and tongues were well above their MIC50 values. VT-1161 was highly effective as treatment of both fluconazole-susceptible and -resistant oropharyngeal candidiasis in Act1-/- mice. Conclusions VT-1129 and VT-1161 exhibit significant in vitro activity against Candida strains, including fluconazole-resistant C. albicans and C. glabrata. VT-1161 administration in mice results in significant mucosal drug accumulation and eradicates infection caused by fluconazole-susceptible and -resistant Candida strains.

The homozygous CX3CR1-M280 mutation impairs human monocyte survival

Several reports have demonstrated that mouse Cx3cr1 signaling promotes monocyte/macrophage survival. In agreement, we previously found that, in a mouse model of systemic candidiasis, genetic deficiency of Cx3cr1 resulted in increased mortality and impaired tissue fungal clearance associated with decreased macrophage survival. We translated this finding by showing that the dysfunctional CX3CR1 variant CX3CR1-M280 was associated with increased risk and worse outcome of human systemic candidiasis. However, the impact of this mutation on human monocyte/macrophage survival is poorly understood. Herein, we hypothesized that CX3CR1-M280 impairs human monocyte survival. We identified WT (CX3CR1-WT/WT), CX3CR1-WT/M280 heterozygous, and CX3CR1-M280/M280 homozygous healthy donors of European descent, and we show that CX3CL1 rescues serum starvation-induced cell death in CX3CR1-WT/WT and CX3CR1-WT/M280 but not in CX3CR1-M280/M280 monocytes. CX3CL1-induced survival of CX3CR1-WT/WT monocytes is mediated via AKT and ERK activation, which are both impaired in CX3CR1-M280/M280 monocytes, associated with decreased blood monocyte counts in CX3CR1-M280/M280 donors at steady state. Instead, CX3CR1-M280/M280 does not affect monocyte CX3CR1 surface expression or innate immune effector functions. Together, we show that homozygocity of the M280 polymorphism in CX3CR1 is a potentially novel population-based genetic factor that influences human monocyte signaling.

Novel signal transducer and activator of transcription 1 mutation disrupts small ubiquitin-related modifier conjugation causing gain of function

Background Sumoylation is a posttranslational reversible modification of cellular proteins through the conjugation of small ubiquitin‐related modifier (SUMO) and comprises an important regulator of protein function. Objective We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1). Methods STAT1 sequencing and functional characterization were performed in transfection experiments by using immunoblotting and immunoprecipitation in STAT1‐deficient cell lines. Transcriptional response and target gene activation were also investigated in PBMCs. Results We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif (702IKTE705) in a patient with disseminated Rhodococcus species infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism, and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, whereas it was present in wild‐type (WT) STAT1 cells, as well as the loss‐of‐function mutants L706S and Y701C. Furthermore, stimulation with IFN‐&ggr; led to enhanced STAT1 phosphorylation, enhanced transcriptional activity, and target gene expression in the E705V‐transfected compared with WT‐transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of the phosphorylation site Y701, which corresponded to the loss‐of‐function and gain‐of‐function variants. Conclusion This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key posttranslational regulatory modification of STAT1 and identify a novel mechanism for gain‐of‐function STAT1 disease in human subjects.

VT-1598 inhibits the in vitro growth of mucosal Candida strains and protects against fluconazole-susceptible and -resistant oral candidiasis in IL-17 signalling-deficient mice

Background Chronic mucocutaneous candidiasis (CMC) treatment often induces drug resistance, posing long-term challenges. A novel broad-spectrum fungal CYP51 inhibitor, VT-1598, specifically targets fungal CYP51, but not human CYP enzymes. Objectives To determine the efficacy of VT-1598 in the treatment of oral Candida infection caused by fluconazole-susceptible and -resistant clinical isolates. Methods The MICs of VT-1598 and fluconazole for 28 Candida isolates recovered from patients with inherited CMC were determined using CLSI M27-A3 and M27-S4 guidelines. Plasma and tongue VT-1598 or fluconazole concentrations were measured in mice following oral administration to determine tissue distribution. Tongue fungal load was determined in IL-17 signalling-deficient Act1-/- mice following sublingual Candida albicans infection and oral treatment with fluconazole or VT-1598. Results Among the 28 Candida isolates, 10 (36%) had fluconazole MICs of ≥4 mg/L, whereas VT-1598 demonstrated potent in vitro activity against all isolates (MIC90, 0.125 mg/L). After oral administration, VT-1598 levels in mouse plasma and tongue were significantly greater than those of fluconazole. In vivo, VT-1598 exhibited significant efficacy against fluconazole-susceptible and -resistant C. albicans, even at low drug doses. Furthermore, after a 10 day washout period, tongue fungal burdens in fluconazole-treated mice returned to vehicle control levels, whereas, in contrast, they were undetectable in mice treated with VT-1598. Conclusions VT-1598 effectively controls in vitro growth of mucosally derived Candida clinical isolates, including fluconazole-resistant strains. In vivo, VT-1598 eliminates C. albicans, even after a long washout period or at low doses. Therefore, VT-1598 is a promising drug candidate that may significantly improve treatment options for CMC patients.

VT-1598 Inhibits the in vitro Growth of Mucosal Candida Isolates and Protects Against Oropharyngeal Candidiasis in IL-17 Deficient Mice

Abstract Background Patients with chronic mucocutaneous candidiasis (CMC) often develop azole-resistant Candida infections, making treatment difficult due to lack of oral antifungal drug options. VT-1598 is a novel broad-spectrum fungal CYP51 inhibitor designed for exquisite selectivity for the fungal target versus human CYP enzymes to circumvent classic azole side effects like drug–drug interactions. We report the efficacy of VT-1598 in the treatment of oral Candida infection (including by azole-resistant strains). Methods The in vitro MIC values of 28 Candida species isolated from patients with CMC due to AIRE mutations were tested against VT-1598 and fluconazole (FLC), using CLSI broth microdilution M27-S4. Plasma VT-1598 levels were measured using LC–MS/MS with electrospray ionization. Tongue fungal load was determined in IL-17 deficient Act1- /- mice following sublingual C. albicans infection and once-daily oral treatment for 4 days with 25 mg/kg FLC or 3.2, 8, and 20 mg/kg VT-1598 starting 18 hours post-infection. Results Among 28 Candida isolates tested (22 C. albicans, three C. glabrata, and one each of C. utilis, C. dubiliensis, and C. krusei), 10 (36%) were not susceptible to FLC, based on CLSI breakpoints (>4 mg/ml). Remarkably, all 28 isolates were highly susceptible to VT-1598 (MIC50 and MIC90, 0.06 and 0.125 mg/ml, respectively). Oral administration of VT-1598 led to mean drug levels in mouse plasma (2.0, 3.0, and 11 mg/ml at the low, mid, and high doses, respectively) that were higher than the MIC values. In vivo, VT-1598 was significantly more effective, compared with FLC, against FLC-susceptible and -resistant C. albicans, and led to elimination of fungal growth even at the lowest tested dose (3.2 mg/kg). After a 10-day washout period from the last dose, mice treated with VT-1598 did not have mucosal fungal growth, while mice treated with FLC had tongue fungal loads similar to vehicle control. Conclusion VT-1598 shows in vitro activity against mucosally derived Candida, including FLC-resistant strains. In vivo, VT-1598 achieves high plasma concentrations and eliminates viable C. albicans, even at low doses and after an extended washout period. These data indicate that VT-1598 may be a significantly improved treatment option for patients with CMC. Disclosures C. M. Yates, Viamet Pharmaceuticals, Inc.: Employee, Salary. O. J. Cohen, Viamet Pharmaceuticals, Inc.: Employee, Salary. R. J. Schotzinger, Viamet Pharmaceuticals, Inc.: Employee, Salary. E. P. Garvey, Viamet Pharmaceuticals, Inc.: Employee, Salary. M. Lionakis, Viamet Pharmaceuticals: Research support (CRADA), Research support