Chloroquine Inhibits Paracoccidioides brasiliensis Survival within Human Monocytes by Limiting the Availability of Intracellular Iron

Abstract

Paracoccidioidomycosis is a deep mycosis that is endemic in Latin America. This disease is caused by Paracoccidioides brasiliensis, a fungus that undergoes thermal dimorphism, developing as yeast at body temperature and as a mycelium at room temperature (16). This fungus causes natural infections by inhalation of conidia or mycelial elements. These conidia convert to the parasitic yeast form in the lungs of mice (39). Ingested conidia or yeast-forms of P. brasiliensis readily multiplied inside murine alveolar or peritoneal macrophages; however, if they were activated by cytokines, such as IFN-γ, the multiplication was limited and conidia or yeast cells were killed (5,10). Similarly, the interaction of human phagocytic cells and yeastform of P. brasiliensis was studied in vitro and yeast cells were readily ingested by monocytes or monocytederived macrophages. However, these cells allowed P. brasiliensis multiplication, unless they were activated with IFN-γ or culture supernatants from concanavalin-A-stimulated mononuclear cells, suggesting that this activation could be responsible for iron restriction and the consequent decrease in the survival of P. brasiliensis Abstract: The mechanisms used by Paracoccidioides brasiliensis (Pb 18) to survive into monocytes are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens, including P. brasiliensis, whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Chloroquine, by virtue of its basic properties, has been shown to prevent release of iron from holotransferrin by raising endocytic and lysosomal pH, and thereby interfering with normal iron metabolism. Then, in view of this, we have studied the effects of CHLOR on P. brasiliensis multiplication in human monocytes and its effect on the murine paracoccidioidomycosis. CHLOR induced human monocytes to kill P. brasiliensis. The effect of CHLOR was reversed by FeNTA, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. CHLOR treatment of Pb 18-infected BALB/c mice significantly reduced the viable fungi recovery from lungs, during three different periods of evaluation, in a dose-dependent manner. This study demonstrates that iron is of critical importance to the survival of P. brasiliensis yeasts within human monocytes and the CHLOR treatment in vitro induces Pb 18 yeast-killing by monocytes by restricting the availability of intracellular iron. Besides, the CHLOR treatment in vivo significantly reduces the number of organisms in the lungs of Pb-infected mice protecting them from several infections. Thus, CHLOR was effective in the treatment of murine paracoccidioidomycosis, suggesting the potential use of this drug in patients' treatment. Chloroquine Inhibits Paracoccidioides brasiliensis

Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

“…In this way, Paracoccidioides has at least two different opportunities to be exposed to the heme group: during (i) fungal dissemination by the hematogenous route or (ii) macrophage infection. Because it has been suggested that monocyte intracellular iron availability is required for Paracoccidioides survival [37], the ability of the rbt5 knockdown strain to survive inside macrophages was investigated. The rbt5 knockdown strain presented decreased survival inside macrophages in comparison with control strains, which indicates that Rbt5 could be a virulence factor and/or could affect macrophage stimulation to kill the internalized yeast cells.…”

“…Possible strategies that are used by Paracoccidioides to survive inside macrophages include (i) the downregulation of macrophage genes that are involved in the inflammatory response and in the activation against pathogens [33], [34], (ii) the inhibition of phagosome-endosome fusion [35] and (iii) the detoxification of ROS that are produced by the phagocyte NADPH oxidase system [36]. Moreover, iron availability inside monocytes is required for Paracoccidioides survival because the effect of chloroquine on fungal survival is reversed by FeNTA, which is an iron compound that is soluble in the neutral to alkaline pH range [37].…”

Successful Treatment of Psoriasis-like Lesions in HIV Infected Patients in Uganda with Chloroquine

“…The lesions of Patient 2 were also suggestive of a generalized fungal skin infection but they did not respond to antifungal therapy. Chloroquine has also been shown to be effective in the treatment of fungal infections caused by Histoplasma capsulatum [43], Paracoccidiodes brasiliensis [44] and Cryptococcus neoformans [45]. This effect is thought to be mediated by reducing the availability of iron for the intracellular pathogen [43,46,47].…”

High expression of human monocyte iNOS mRNA induced by Paracoccidioides brasiliensis is not associated with increase in NO production

“…Yeast viability was determined [19] and suspensions containing more than 90% viable yeasts were used.…”

“…The inoculum used for the challenge was also plated according to the same conditions. Then, the plates containing the product of macrophageefungus cocultures were considered as experimental plates and those plated with the inoculum alone, were used as controls [19]. The fungicidal activity percentage was determined by the following formula: % Fungicidal Activity ¼½1 À ðmean CFU recovered on experimental plates=mean CFU recovered on control platesÞ Â 100 NO production was quantified in cocultures, by the accumulation of nitrite in the supernatants using the standard Griess assay [20].…”