We investigated the role of intracellular iron on the capacity of Histoplasma capsulatum (Hc) yeasts to multiply within human macrophages (Mphi). Coculture of Hc-infected Mphi with the iron chelator deferoxamine suppressed the growth of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by iron-free transferrin (apotransferrin). Chloroquine, which prevents release of iron from transferrin by raising endocytic and lysosomal pH, induced human Mphi to kill Hc. The effect of chloroquine was reversed by iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. Chloroquine (40-120 mg/kg) given intraperitoneally for 6 d to Hc-infected C57BL/6 mice significantly reduced the growth of Hc in a dose-dependent manner. At 120 mg/kg there was a 17- and 15-fold reduction (P < 0.01) in CFU in spleens and livers, respectively. The therapeutic effect of chloroquine also correlated with the length of treatment. As little as 2 d of chloroquine therapy (120 mg/kg), when started at day 5 after infection, reduced CFU in the spleen by 50%. Treatment with chloroquine for 10 d after a lethal inoculum of Hc protected six of nine mice; all control mice were dead by day 11 (P = 0.009). This study demonstrates that: (a) iron is of critical importance to the survival and multiplication of Hc yeasts in human Mphi; (b) in vitro, chloroquine induces Mphi killing of Hc yeasts by restricting the availability of intracellular iron; and (c) in vivo, chloroquine significantly reduces the number of organisms in the spleens and livers of Hc-infected mice and can protect mice from a lethal inoculum of Hc yeasts. Thus, chloroquine may be effective in the treatment of active histoplasmosis and also may be useful in preventing relapse of histoplasmosis in patients with acquired immunodeficiency syndromes.

/pdf/chloroquine-induces-human-macrophage-killing-of-histoplasma-oacus497kz.pdf

Chloroquine induces human macrophage killing of Histoplasma capsulatum by limiting the availability of intracellular iron and is therapeutic in a murine model of histoplasmosis.

SUMMARY:
The interaction of human monocytes or monocyte-derived macrophages and yeast-form Paracoccidioides brasiliensis was studied in vitro. Yeast cells were readily ingested by adherent monocytes or macrophages. Multiplication of P. brasiliensis, measured by growth as colony forming units (cfu) on a supplemented medium with good plating efficiency, was greater in monocyte co-cultures compared to the number of cfu obtained from complete tissue-culture medium (CTCM). Multiplication increased with time in macrophage co-cultures, e.g., from two-six-fold in 24 h to nine-fold in 72 h. Microscopic observations indicated that ingested yeast cells multiplied inside macrophages. When monocytes were treated with supernate cytokines (CK) from concanavalin-A-stimulated mononuclear cells, then co-cultured with P. brasiliensis, multiplication was significantly inhibited compared with control monocyte co-cultures. Treatment of macrophages—derived from monocytes by culture in vitro for 3 days—for a further 3 days with CK resulted in maximal inhibition of multiplication over the subsequent 72 h. Similarly, when monocyte-derived macrophages (after culture for 7 days) were treated for 3 days with recombinant human γ-interferon (IFN; 300U/ml) or CK they restricted multiplication of P. brasiliensis by 65% and 95%, respectively, compared with control macrophages. Antibody to IFN abrogated the effect of IFN or CK treatment. These findings show that ingested P. brasiliensis can multiply in human monocytes or macrophages and that this multiplication can be restricted by activated monocytes or macrophages.

/pdf/support-of-paracoccidioides-brasiliensis-multiplication-by-fn71xq5o6s.pdf

Support of Paracoccidioides brasiliensis multiplication by human monocytes or macrophages: inhibition by activated phagocytes

Recombinant cytokines and colony-stimulating factors (CSFs) were tested for their abilities to activate human monocytes/macrophages (M phi) to inhibit the intracellular growth of or kill Histoplasma capsulatum yeasts. None of the cytokines or CSFs or combinations of cytokines and CSFs activated M phi fungistatic activity when they were added to M phi monolayers concurrently with yeasts. In contrast, culture of monocytes for 7 days in the presence of interleukin 3, granulocyte-M phi CSF, or M phi CSF stimulated M phi fungistatic (but not fungicidal) activity against H. capsulatum yeasts in a concentration-dependent manner. Optimal activation of M phi by CSFs required 5 days of coculture, and the cultures had to be initiated with freshly isolated peripheral blood monocytes. Culture of monocytes with combinations of CSFs or addition of CSFs during the 24 h of coculture with the yeasts did not further enhance M phi fungistatic activity for H. capsulatum. Addition of gamma interferon or tumor necrosis factor alpha to CSF-activated M phi also did not enhance M phi fungistatic activity. These results suggest that interleukin 3, granulocyte-M phi CSF, and M phi CSF may play a role in the cell-mediated immune response to H. capsulatum by enhancing monocyte/M phi fungistatic activity.

https://iai.asm.org/content/iai/60/11/4593.full.pdf

Colony-stimulating factors activate human macrophages to inhibit intracellular growth of Histoplasma capsulatum yeasts.

Human neutrophils (PMN) demonstrated potent fungistatic activity against Histoplasma capsulatum (Hc) yeasts in a sensitive microassay that quantifies the growth of yeasts by the incorporation of [3H]leucine. At a PMN:yeast ratio of 1:2, PMN inhibited the growth of yeasts by 37%. Maximum inhibition of 85% to 95% was achieved at a PMN/yeast ratio of 10:1 to 50:1. Opsonization of the yeasts in fresh or heat-inactivated serum was required for PMN-mediated fungistasis, but ingestion of the yeasts was not required. Recognition and phagocytosis of opsonized yeasts was via PMN complement receptor (CR) type 1 (CR1), CR3, and FcRIII (CD16). PMN fungistatic activity was evident by 2 h, was maximum at 24 h, and persisted up to 5 d. In contrast, yeasts multiplied within monocytes to a greater extent than in culture medium alone. PMN from three patients with chronic granulomatous disease (CGD) inhibited the growth of Hc yeasts by an average of 97%, compared with 86% in three normal controls. Furthermore, preincubation of PMN with the lysosomotropic agent NH4Cl inhibited fungistatic activity in a concentration-dependent manner. Finally, experiments with subcellular fractions of PMN demonstrated that the principal component of the fungistatic activity of PMN was localized in the azurophil granules. These data demonstrate that human PMN possess potent fungistatic activity against Hc yeasts and further show that fungistasis is mediated by antimicrobial agents contained in the azurophil granules.

/pdf/human-neutrophil-mediated-fungistasis-against-histoplasma-4d59p0hh0p.pdf

Human neutrophil-mediated fungistasis against Histoplasma capsulatum. Localization of fungistatic activity to the azurophil granules.

Infection with Histoplasma capsulatum results in a subclinical infection in immunocompetent hosts due to an effective cellular immune response. By contrast, immunodeficient individuals can have a severe disseminated and potentially fatal disease. In a previous study, it was demonstrated that normal mice infected intravenously with H. capsulatum and treated with interleukin-12 (IL-12) at the time of infection were protected from a fatal outcome. In this study, we examined the immunomodulatory effects of IL-12 on disseminated histoplasmosis in immunodeficient SCID mice. SCID mice infected with H. capsulatum and treated with IL-12 showed an increase in survival and a reduction in the colony counts of H. capsulatum in internal organs at 14 days after infection. The protective effect of IL-12 was abrogated if animals were also treated with a neutralizing antibody to gamma interferon (IFN-gamma). IL-12 treatment also resulted in an increase in mRNA expression and protein production for IFN-gamma, tumor necrosis factor alpha (TNF-alpha), and nitric oxide from spleen cells. When IL-12 was combined with amphotericin B (AmB) treatment, there was a significant increase in survival compared with either modality alone. Moreover, combined treatment resulted in an increase in both IFN-gamma and TNF-alpha production, as well as in a substantial reduction in H. capsulatum burden at 35 and 90 days postinfection. This study demonstrates that IL-12 modulates the protective immune response to histoplasmosis in SCID mice and also suggests that IL-12 in combination with AmB may be useful as a treatment for H. capsulatum in immunodeficient hosts.

Interleukin-12 modulates the protective immune response in SCID mice infected with Histoplasma capsulatum.

The interaction of human macrophages with the yeast form of the thermally dimorphic fungal pathogen, Histoplasma capsulatum, was studied. Macrophages derived from monocytes by culture in vitro for 3 days ingested H. capsulatum, but were neither fungicidal or fungistatic. In contrast, when monocytes were exposed to human recombinant gamma-interferon (gamma-IFN) during their differentiation into macrophages, those macrophages were able to reduce the number of ingested or adherent cfu of H. capsulatum by 44-75% in 2 h. Activation of macrophages for fungicidal activity by gamma-IFN was dose dependent and 500-1000 units ml were optimal. Antibody to gamma-IFN abrogated the gamma-IFN activation process. Killing of H. capsulatum by activated macrophages in 2-h assays could be inhibited by superoxide dismutase but not by sodium azide.

Killing of Histoplasma capsulatum by γ-interferon-activated human monocyte-derived macrophages: evidence for a superoxide anion-dependent mechanism

Murine polymorphonuclear neutrophils (PMN) were examined for fungicidal and fungistatic activity against yeast cells of Histoplasma capsulatum. In a 16-h limiting dilution assay (LDA) (constant number of PMN versus decreasing numbers of yeast cells) where PMN to yeast cell ratios were high (0.4-1.5 x 10(5):1), murine PMN exhibited a limited fungicidal effect on H. capsulatum as measured by sterilization of cultures containing one to four yeast cells. On the other hand, inoculum colony forming units (c.f.u.) of H. capsulatum were not reduced by PMN in short-term (2 h) assays (PMN to yeast cell ratio, 500:1) even though H. capsulatum stimulated a brisk oxidative burst in PMN. In the same assays 84% of Candida albicans cells were killed. These findings indicate that H. capsulatum was resistant to killing by products of the oxidative burst generated by PMN and that it was slightly sensitive to PMN in 16-h LDA assays where a different microbicidal mechanism may be operative. In long-term co-culture assays, where the PMN to yeast cell ratio was 500:1, PMN consistently inhibited the replication of H. capsulatum after 24, 48, or 72 h of incubation. Inoculum c.f.u. failed to increase significantly in co-cultures, whereas in culture medium alone c.f.u. increased several-fold in 72 h. Based on these novel findings, we speculate that PMN might play a role in resistance to H. capsulatum due to their strong fungistatic activity and to a lesser extent their limited fungicidal action.

Antifungal activity of murine polymorphonuclear neutrophils against Histoplasma capsulatum.

The mechanism by which the yeast form of Blastomyces dermatitidis resists killing by human peripheral blood polymorphonuclear neutrophils (PMN) was investigated. The metabolic products of the oxidative burst generated during the interaction of PMN and B. dermatitidis or Candida albicans were detected by lucigenin- or luminol-enhanced chemiluminescence (CL). Interaction of PMN and C. albicans resulted in luminol-enhanced CL 100-fold greater than that generated by PMN and B. dermatitidis. This correlated with killing of C. albicans and resistance of B. dermatitidis. Since B. dermatitidis and PMN interactions resulted in significant lucigenin-enhanced CL, deficient luminol CL was not due to a lack of products from the NADPH oxidase system. Killed B. dermatitidis cells at 37°C were more efficient than live cells in stimulating PMN for luminol-enhanced CL; however, only fragmented B. dermatitidis cells elicited luminol-enhanced CL equivalent to that of C. albicans. Since lysates of PMN were active in a cell-fr...

A basis for resistance of blastomyces dermatidis killing by human neutrophils: inefficient generation of myeloperoxidase system products

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Killing of Histoplasma capsulatum by γ-interferon-activated human monocyte-derived macrophages: evidence for a superoxide anion-dependent mechanism

Antifungal activity of murine polymorphonuclear neutrophils against Histoplasma capsulatum.

A basis for resistance of blastomyces dermatidis killing by human neutrophils: inefficient generation of myeloperoxidase system products