Treatment with pCramoll Alone and in Combination with Fluconazole Provides Therapeutic Benefits in C. gattii Infected Mice

Abstract

Cryptococcus gattii is one of the main causative agents of cryptococcosis in immunocompetent individuals. Treatment of the infection is based on the use of antimycotics, however, the toxicity of these drugs and the increase of drug-resistant strains have driven the search for more effective and less toxic therapies for cryptococcosis. pCramoll are isolectins purified from seeds of Cratylia mollis, a native forage plant from Brazil, which has become a versatile tool for biomedical application. We evaluated the effect of pCramoll alone and in combination with fluconazole for the treatment of mice infected with C. gatti. pCramoll alone or in combination with fluconazole increased the survival, reduced the morbidity and improved mice behavior i.e., neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength and reflex/sensory function. These results were associated with (i) decreased pulmonary and cerebral fungal burden and (ii) increased inflammatory infiltrate and modulatory of IFNγ, IL-6, IL-10, and IL-17A cytokines in mice treated with pCramoll. Indeed, bone marrow-derived macrophages pulsed with pCramoll had increased ability to engulf C. gattii, with an enhanced production of reactive oxygen species and decrease of intracellular fungal proliferation. These findings point toward the use of pCramoll in combination with fluconazole as a viable, alternative therapy for cryptococcosis management.

Keywords: Cratylia mollis lectin; cryptococcosis; fluconazole; immunomodulation; lectins; survival.

Figure 1

Immunomodulatory and fungicidal effect of pCramoll in murine bone marrow macrophages infected with Cryptococcus gattii. (A) After 3 h of incubation, the phagocytic index increased under pCramoll treatment to 1 μM; (B) After 3 h of incubation, treatment with pCramoll enhances ROS production in macrophages infected with C. gattii, in a concentration-dependent manner. (C) Oxide Nitric production after 3 h after incubation; (D) Intracellular Proliferation Rate after 24 h of incubation; (E) Macrophage morphology after 24 h of incubation with C. gattii, showing C. gattii phagocytosis (arrows). Cell cultures were observed under 100 × optical zoom and 10 fields per coverslip. (F) Area of macrophages stimulated with lectins and subsequently infected with C. gatti. *p < 0.05; ^**p < 0.01; ^***p < 0.005 (Difference between treatment groups and infected control); Φ, Uninfected macrophages, ΦCg, C. gattii-infected macrophages; ΦpC, Uninfected macrophages treated with pCramoll; C+ 10 μM Hydrogen peroxide. Data represent the means ± SE from two independent experiments consisting of ten replicate assays.

Figure 2

Survival curve and behavioral profile evaluation of mice infected with Crytococcus gattii and subjected to different treatments. (A) Six mice per group were inoculated with 10⁴ cells of L27/01 strain by intratracheal line inoculation and treated with fluconazole at 20 mg.kg⁻¹ (FCZ), pCramoll at 1 ug (pC1), or the combination (pC1 + FCZ). Animals treated by pC1 + FCZ had an average increase of over 34.8% in survival compared to those treated with FCZ alone ^*p < 0.05. (B–G) Five animals per group were submitted to the SHIRPA Protocol. There was no behavioral difference between uninfected mice (NI) and those treated with pC1 alone, which did also not influence the survival of mice. Untreated mice (NT); ^*p < 0.05; ^**p < 0.001 (difference when compared to NI); ⁺p < 0.5; ⁺⁺p < 0.001 (difference when compared to NT); ^#p < 0.05 (difference between FCZ and combination). Data represent the means ± SE from three independent experiments.

Figure 3

pCramoll in combination with fluconazole shows increased survival and reduced fungal burden in the lungs and brain of Cryptococcus gattii-infected mice. Six mice per group were inoculated with 10⁴ cells of the L27/01 strain by intratracheal line inoculation and given the combination treatment, analyzed at 15 and 35 dpi: (A,B) Colony-forming Units (CFU) recovered from brain and lungs, respectively, at 15 and 35 days post infection; (C) Histological sections of lung tissue stained with H&E at 200 ×, 15 days post inoculation. Non-infected mice showed normal histology. Mice treated with pC1 showed more prominent perivascular inflammatory infiltrate (thin arrow) and less yeasts (thick arrow) in alveolar space than untreated, infected mice. Mice from FLZ or pC1+ FLZ showed moderate inflammation. Uninfected animals (NI), infected untreated control (NT), groups treated with pCramoll at 1 μg (pC1), Fluconazole (FCZ), ND (not recovered CFU). ^*p < 0.05; ^**p < 0.01 (difference between treated groups and NT); ^#p < 0.05 (difference between FCZ alone and combination, at 35 dpi). Data represent the means ± SE from three independent experiments consisting of triplicate assays.

Figure 4

Inflammatory response of pCramoll in combination with fluconazole in Cryptococcus gattii-infected mice, analyzed at 15 and 35 dpi: (A,B) Total and differential count of leukocytes (neutrophils and mononuclear cells) in the bronchoalveolar lavage, respectively; at 15 and 35 days after infection; (C,D) Myeloperoxidase and N-acetylglucosaminidase activities in lungs of infected animals; (E–H) Levels of cytokines IFN-γ, IL-6, IL-10, and IL-17A in lungs. Uninfected animals (NI), infected untreated control (NT), groups treated with pCramoll at 1 μg (pC1), Fluconazole (FCZ), ^*p < 0.05; ^**p < 0.01 (difference between treated groups and NT); ^#p < 0.05 (difference when comparing treated groups); ^###p < 0.005 (difference between FCZ alone and combination, at 35 dpi); ⁺p < 0.5 (difference between 15 dpi and 35 dpi). Data represent the means ± SE from three independent experiments consisting of triplicate assays.