Bionized Nanoferrite Particles Alter the Course of Experimental Cryptococcus neoformans Pneumonia

ABSTRACT

Cryptococcosis is a devastating fungal disease associated with high morbidity and mortality even when treated with antifungal drugs. Bionized nanoferrite (BNF) nanoparticles are powerful immunomodulators, but their efficacy for infectious diseases has not been investigated. Administration of BNF nanoparticles to mice with experimental cryptococcal pneumonia altered the outcome of infection in a dose response manner as measured by CFU and survival. The protective effects were higher at lower doses, with reductions in IL-2, IL-4, and TNF-α, consistent with immune modulation whereby reductions in inflammation translate into reduced host damage, clearance of infection, and longer survival.

INTRODUCTION

Cryptococcus neoformans is a fungal pathogen with a worldwide distribution. Serological studies of human populations show a high prevalence of human infection, which rarely progresses to disease in immunocompetent hosts (1, 2). However, decreased host immunity places individuals at high risk for cryptococcal disease. The disease can result from acute infection or reactivation of latent infection, in which yeasts within granulomas and host macrophages emerge to cause disease (3). In both immunocompetent and immunosuppressed patients, cryptococcosis has high morbidity and mortality, even with aggressive antifungal drug therapy (1). Current therapies can require antifungal drugs for several months, and treatment can be complicated by rising antimicrobial drug resistance (4, 5). Consequently, development of new therapeutics against C. neoformans infections is urgently required.

Magnetic iron oxide nanoparticles (MNPs) have proven useful for the diagnosis and therapy of various conditions because they display generally favorable biocompatibility and varied responsiveness to magnetic fields (6–10). When injected into the bloodstream, nanoparticles encounter a complex fluid environment that can modify the initial particle surface to one having a molecular signature that produces specific interactions with host biology (11–13). Ultimately, although indirectly, the interactions of nanoparticles with the complex host environment can lead to their deposition in organs or tissues that depends upon the initial physical and chemical properties of the injected construct (14). The immune system's function in the maintenance of tissue homeostasis is to protect the host from environmental agents such as microbes or chemicals, and thereby preserve the integrity of the body. The study of the interactions between nanoparticles and various components of the immune system is an active area of research in bio- and nanotechnology because the benefits of using nanotechnology in industry and medicine are often questioned over concerns regarding the safety of these novel materials (11, 15, 16). The last decade of research has shown that, while in certain situations nanoparticles can be toxic, nanotechnology engineering can modify these materials to either avoid or target the immune system. Depending on the nanoparticle composition and physicochemical properties, nanoparticle interactions with host biology can induce or increase inflammation (17, 18) or mediate immunosuppression (19, 20). Either of these responses can be used to enhance efficacy in specific disease contexts, and which response is desired depends on specific features of both the disease and nanoparticle. For a nanoparticle to induce both inflammation and immunosuppression in the same disease context, however, is unusual. Specific targeting of the immune system, on the other hand, provides an attractive option for vaccine delivery, as well as for improving the quality of anti-inflammatory, anticancer, and antiviral therapies (21–24). Macrophages utilize multiple routes to ingest the same types of nanoparticles (25). Several studies reported that smaller particles (20–200 nm) elicit stronger immune responses than their larger counterparts (11, 26–29).

Previous studies have shown that injection of nanoparticles into experimental animals elicited pseudo infection-like response or local inflammation or systemic immune response leading to immunological changes in tumor microenvironment in mouse models of cancer (30, 31). When delivered systemically, BNF nanoparticles can trigger an immune response that resembles infection, leading to downstream signaling that engages effector antitumor CD8⁺ T cell function (31). The effect of nanoparticles on tumor biology has attracted considerable attention, but there has been no comparable effort to study their effect on infectious diseases. Like other engineered nanoparticles (NPs), BNF NPs inherently possess physical features resembling viruses and elicit an antitumor immune response when injected into immunocompetent mouse models of breast cancer (31).

In this study we tested the effect of BNF in a mouse model of cryptococcal pneumonia. Our results show that BNF nanoparticles have a profound effect on the inflammatory response to C. neoformans infection that is associated with altered outcomes depending on the nanoparticle dose used.

RESULTS

The fungal burden was assessed from the removed lungs, and mice treated with BNF at high dose had higher CFU (Fig. 1A); CFU numbers between groups were not statistically significant, but there was a trend toward higher fungal burden in the BNF-treated groups, with higher numbers in groups that received BNF 24 h after the challenge with C. neoformans (Fig. 1A). Histological slides of lungs showed that PBS-treated group had some inflammation (Fig. 1B), whereas nanoparticle injection (5 mg Fe/mouse) before (Fig. 1C) or after 24 h (Fig. 1D) of infection had increased inflammation seen by hematoxylin and eosin (H&E). It is possible to observe in the slides an increased number of yeasts in both nanoparticle-treated groups (Fig. 1F and G) compared to control (Fig. 1E), as shown by periodic acid-Schiff stain (PAS) of lungs. This aggravated reaction could be due to the very high concentration of nanoparticle used in this initial study.

FIG 1.

Higher recovery of lungs CFU 60 days after challenge with C. neoformans strain 24067 from the BNF-treated groups compared with the PBS control group (A). Intense immune reaction with numerous yeast organisms was seen following treatment with higher concentrations of nanoparticles in FVB/NJ mice. Representative histology images after 60 days of infection in PBS treated group (B), and nanoparticle injection (5 mg Fe/mouse) before 24 h (C) or after 24 h (D) by H&E. It is possible to observe an increased amount of yeast in nanoparticle treated groups (F, G) compared to control (E), by PAS. Black arrows in E-G indicate yeast, red arrows indicate foamy macrophages, and yellow indicates multinucleated giant cells. Blue arrow in F indicate organism inside a macrophage, and green indicates neutrophils.

Given the results from the high dose BNF experiment in FVB/N mice, we evaluated whether lower doses of BNF nanoparticles in the same model of FVB/N mice would produce different outcomes. A significant decrease (P < 0.05) of fungal CFU from the recovered lungs was observed in mice treated with lowest dose of BNF (0.005 mg) (Fig. 2A), demonstrating that at lower doses, BNF NPs (as 0.005 mg) could protect against C. neoformans infection. Corroborating with CFU data, H&E-stained lung sections show inflammatory cells surrounding Cryptococcus yeast in FVB/NJ mice treated with PBS, with numerous macrophages and multinucleated giant cells (blue arrow) and neutrophils (green arrow; Fig. 2B and C). BNF (0.005 mg) treated lungs had fewer inflammatory cells (Fig. 2D and 2E). PAS staining showed presence of multiple fungal yeasts in PBS treated mice (Fig. 2F), whereas lower concentration (BNF 0.005 mg) of nanoparticle treated lungs showed no positive staining for yeast (Fig. 2G). Prussian blue staining for nanoparticles in lungs also showed no evidence of iron (BNF) deposits (data not shown).

FIG 2.

BNF 0.005 mg doses showed a significant decrease (P < 0.05) of fungal CFU from recovered lungs (A), indicating a protective profile in lungs 14 days after challenge with C. neoformans strain 24067. H&E-stained lung in FVB/NJ mice treated with PBS showing numerous macrophages and multinucleated giant cells (blue arrow) and neutrophils (green arrow) (B, C). BNF (0.005 mg) treated lungs (D, E). PAS staining in PBS treated mouse (F) and in nanoparticle treated lungs (G). Tissues were stained with monoclonal antibody 18B7 conjugated with Oregon green and counterstained with DAPI. Control PBS (H), BNF 0.5 mg (I), BNF 0.05 mg (J), BNF 0.005 mg (K) ×10 magnification. Representative graph showing the IF score generated by the IF slides (L). Black arrows show fungal yeast cells. *, P < 0.05.

To further analyze the protective effect observed in FVB/N mice treated with lower dose of BNF, we carried out a survival study in A/J mice. A/J mice were chosen to test whether the high innate resistance of FVB/N mice to cryptococcal infection, which survived a high C. neoformans inoculum such that none died after 60 days, was responsible for the observed effects. In this experiment, we analyzed both mouse survival and lung fungal burden in different groups. Mice receiving the lowest dose of BNF (0.0005 mg) showed a significant increase (P < 0.05) in the survival, with no deaths (survival of 100%) after 60 days, in comparison with the control group (PBS injected), with a survival of only 20% (Fig. 3A). To analyze the fungal burden, lungs were removed 14 days after infection and CFU numbers were determined. Similarly, a significant decrease (P < 0.05) in the number of recovered lung CFUs was observed in the group that received the BNF at 0.0005 mg (Fig. 3B). These data suggest that at lower dose, systemic treatment with BNF NPs can stimulate a protective response when challenged by two different strains of C. neoformans in two different mouse strains. We observed nearly complete remission of disease following a single treatment with low dose BNF NPs in A/J mice. Analysis of H&E-stained lung sections revealed numerous and varied inflammatory cells surrounding Cryptococcus yeast indicative of a robust and organized immune response (blue arrow) (Fig. 3C and D). Additionally, BNF nanoparticle (0.0005 mg Fe/mouse) treated mouse lungs had fewer inflammatory cells in lungs (Fig. 3E and 3F). PAS staining showed presence of numerous Cryptococcus in PBS treated control mouse lungs (Fig. 3G, black arrow). BNF nanoparticle (0.0005 mg Fe) treated mouse lungs showed no positive staining for yeast (H). We noted less MAb 18B7 immunofluorescence in mouse tissues treated with low dose nanoparticles (0.0005 mg) (Fig. 3K and 3L). Mice treated with higher nanoparticle doses (0.005 mg) (Fig. 3J and 3L) had C. neoformans similar to that of the control group (Fig. 3I and 3L). In the lungs and spleens of mice given the lower doses of BNF, the levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) was decreased after 14 days of infection of the BNF 0.005 mg group relative to control mice infected with C. neoformans (P < 0.05) (Fig. 3M). Additionally, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon-gamma (IFN-γ) were analyzed, but no statistical differences were observed between the groups (Fig. 3M). We focused on the low BNF doses since these were associated with better control of infection.

FIG 3.

A/J mice injected with lower doses of BNF showed a protective profile in lungs 14 days after challenge with C. neoformans strain H99. Mice injected with the lower doses of BNF nanoparticles (0.0005 mg) showed a survival of 100% in comparison with the control group (no BNF nanoparticle injection) with a survival of only 20% (P < 0.05) (A). Lung fungal burden showed a significant decrease (P < 0.05) in the number of recovered CFU in the mice that received the BNF 0.0005 mg (B). H&E-stained lung sections showed numerous inflammatory cells surrounding Cryptococcus yeast (blue arrow) (C, D). BNF nanoparticle (0.0005 mg Fe/mouse) treated mouse (E, F). PAS staining showed presence of numerous Cryptococcus in PBS treated control mouse lung (G, black arrow). BNF nanoparticle (0.0005 mg Fe) treated (H). Tissues were stained with monoclonal antibody 18B7 conjugated with Oregon green with DAPI (×10 magnification). Control PBS (I), BNF 0.005 mg (J), and BNF 0.0005 mg (K). Representative graph showing the IF score generated by the IF slides (L). Lung and spleen cytokine levels during the course of infection (M). *, P < 0.05.

Lab culture passages can affect the pathogenicity of cultured Cryptococcus strains (32). To test the potential effects of lab strain variability, we thawed a second vial of strain H99 to infect mice in Study IV to compare the effects with assays of fungal burden and cytokines from lung and spleen in A/J mice. In contrast to results obtained from Study III (Fig. 3B), there was no statistically significant difference in the number of recovered CFU measured in lungs between PBS control and BNF 0.0005 mg groups (Fig. 4A). There was, however, a slight decrease in the CFU number measured in the BNF 0.0005 mg Fe group from spleen, but this was not statistically significant (Fig. 4A). From the lung and spleen cytokines analysis, levels of proinflammatory cytokines, including interleukin-2 (IL-2), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), were decreased after 14 days of infection relative to control mice infected with C. neoformans (Fig. 4B), but this difference was statistically significant only for IL-2 (P < 0.005). Interleukin-10 (IL-10) and interferon-gamma (IFN-γ) were analyzed by no statistically significant differences were observed between both groups (Fig. 4B).

FIG 4.

Fungal burden from lungs and spleens of A/J mice 14 days after challenge with recently thawed C. neoformans strain H99 showed a slight decrease from spleens of BNF 0.0005 mg group compared with PBS control groups (A). Lung and spleen cytokine levels were also analyzed (B).

Table 1 summarizes all the parameters accessed and analyzed between Study I and Study IV. The four independent experiments reported here each differs in several variables, and they represent our experience as we modified the conditions sequentially in an effort to find a set of parameters that resulted in benefit to the mice. Clearly, changes in such variables as mouse background, inoculum, cryptococcal strain, and BNF dose can each have large effects on the outcome of individual experiments. Hence, we caution about interpretations from individual experiments and instead stress that the common result across all the experiments was that BNF administration affected the outcome of C. neoformans infection in mice. These exploratory studies provide encouragement for future additional work to delineate the conditions where BNF particles can be used to modify the outcome of infection.

TABLE 1.

Summary of all the parameters from Study I, Study II, Study III and Study IV^a

Expt	BNF doses	Mice strain	No. of mice	Fungal strain	Time of mice infection	Parameters assessed	CFU data (lung)	CFU data (spleen)	Survival curve (60 days)	Histology (lung)	IF (lung)	IL-2	IL-4	IL-10	IFN-γ	TNF-α
Study I	5 mg (24 h after and before fungal challenge)	FVB/N	5 per group	24067	60 days	CFU, histology (H&E, PAS)	No difference between groups	X	No difference between groups	No difference between groups	X	X	X	HX	X	X
Study II	0.5/0.05/0.005 mg	FVB/N	5 per group	24067	14 days	CFU, histology (H&E, PAS, IF)	Lower CFU no. on 0.005 mg (P < 0.05)	X	X	Less yeasts and inflammation on 0.0005 mg mice group	Less yeasts on 0.0005 mg mice group	X	X	X	X	X
Study III	0.005/0.0005 mg	A/J	10 per group	H99	14 days to sacrifice/60 days survival	CFU, histology (H&E, PAS, IF), survival, cytokine	Lower CFU no. on 0.0005 mg (P < 0.05)	X	Higher survival on both 0.005 & 0.0005 mg groups, (P < 0.05 on 0.0005 mg group)	Less yeasts and inflammation on 0.0005 mg mice group	Less yeasts on 0.0005 mg mice group	Tendency to decrease (lung)	Tendency to decrease (lung)	No difference	Tendency to decrease (lung and spleen)	Decreased (lung and spleen) on 0 0.005 mg group (P < 0.005)
Study IV	0.0005 mg	A/J	10 per group	H99	14 days	CFU, cytokine	No difference between groups	Lower CFU no. on 0.0005 mg	X	X	X	Decreased (lung and spleen) on 0.0005 mg group (P < 0.005)	Tendency to decrease (lung and spleen)	No difference	Tendency to decrease (lung and spleen)	Tendency to decrease (lung and spleen)

^aBNF, bionized nanoferrite; CFU, colony-forming unit; H&E, hematoxylin and eosin; PAS, periodic acid-Schiff; IF, immunofluorescence; IL, interleukin; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon gamma.

DISCUSSION

Clinically, Cryptococcosis is most prevalent in immunocompromised patients, but a significant proportion of cases occur in patients with no apparent immune deficit. One of the confounding paradoxes of this disease is that the prognosis is better in patients with AIDS, possibly because host damage is a function of both fungal processes such as increased intracranial pressure and also tissue damaging effects inflammation (reviewed in reference 33). Consequently, a common denominator in human cryptococcosis, whether it occurs in immunosuppressed or immunologically intact individuals, is dysregulated inflammatory responses, that are ineffective to contain the infection. Since deficits in immune function appear to be major contributors to the outcome of cryptococcosis, it is reasonable to posit that future improvements in therapy will require immune modulators, rather than immune stimulating or immune suppressing treatments (34). In the present study, BNF nanoparticles altered the outcome of infection in a dose response manner, with the best effects observed with low nanoparticle doses, consistent with downregulation of the inflammatory response, possibly associated with immune modulation. Higher doses of BNF particles were associated with stronger inflammation and a trend toward higher fungal burden, suggesting they triggered exuberant immune responses that aggravated infection and damaged host tissue. The association of stronger inflammatory response with lower efficacy is consistent with emerging evidence that host damage in cryptococcosis can have a strong immune mediated component (33).

In summary, our results show that administration of BNF nanoparticles before and after experimental cryptococcal pneumonia infection in mice alters the outcome of infection. Taken together, these preliminary results indicate that iron oxide nanoparticles may harbor immune modulating capabilities to increase/induce and to suppress/reduce inflammation, depending on dose. In this context, nanoparticles comprise important characteristics that make them attractive for a variety of biomedical applications (35). Specifically, iron oxide nanoparticles are physically and chemically stable, biocompatible, and environmentally safe (35), thus presenting unique characteristics for clinical applications. To our knowledge, this is the first exploration of BNF nanoparticles in an infectious disease model, and our results establish the proof of principle that BNF nanoparticles can affect the course of infectious diseases in a manner similar to the experience with cancer and inflammatory diseases. Our results encourage further studies to determine whether they can be used for immunotherapy of cryptococcosis as an adjuvant of standard antifungal therapy.

MATERIALS AND METHODS

C. neoformans serotype D strain 24067 was used in all FVB/N mouse experiments. C. neoformans var. grubii serotype A strain H99 (ATCC 208821) was used in all A/J mice. The yeast cells were kept frozen in 10% glycerol. Sabouraud dextrose broth (SAB, from Gibco) medium was used for standard growth of yeast cells at 30°C with moderate shaking (120 rpm) overnight.

BNF NPs are commercially available as aqueous suspensions of hydroxyethyl starch-coated magnetite (Fe₃O₄) core-shell nanoparticles (Micromod Partikeltechnologie, GmbH, Rostock, Germany). Their synthesis and physical and biological characterization have been extensively documented by us (31, 36–39). BNF NPs are synthesized by precipitating ferric and ferrous sulfate salts from solution at high pH in a high-pressure-homogenization reaction vessel. According to the manufacturer, they have a mean hydrodynamic diameter of ∼100 nm (polydispersity index < 0.25); approximately neutral zetapotential (∼–2 mV @ pH = 7.4); and, Fe content >50% wt/wt (or iron oxide >70% wt/wt) (31).

All animal procedures were performed with prior approval from Johns Hopkins University (JHU) Animal Care and Use Committee (IACUC), under approved protocol number MO18H152. JHU Animal Welfare Assurance Number is D16-00173 (A3272-01). Two mouse strains were used to perform the experiments: 6-week-old female A/J mice (from Jackson Laboratory; JAX stock #000646) and 6- to 8-week-old female FVB/N (from Jackson Laboratory; JAX stock #001800). A/J mice were used because they are highly susceptible to cryptococcal infection (40), whereas our use of FVB/N mice was motivated by our previously reported findings of anticancer immune modulation by BNF NPs in this mouse strain (31). Four different murine experiments were performed as described below. In all experiments, each mouse received a single intravenous injection of either PBS (150 μl/mouse) or BNF at the described dose, according to its group assignment as follows.

Experiment I: Fifteen female FVB/N mice were divided into three groups (n = 5 animals per group): Group 1, PBS 24 h after C. neoformans infection (control PBS); Group 2, BNF 5 mg Fe in 150 μl/mouse 24 h after C. neoformans infection (BNF 5 mg after CN); and Group 3, BNF 5 mg Fe in 150 μl/mouse 24 h before C. neoformans infection (BNF 5 mg before CN). Sixty days after infection, surviving animals were euthanized and tissues extracted for fungal burden and histology analysis.

Experiment II: Experimental design was modeled from Study I, but with de-escalating BNF dose to determine dose-response. Analysis of Study I results indicated potent immune responses, motivating a dose de-escalation study. Another 20 female FVB/N mice were divided into four groups (n = 5 animals per group), representing PBS control and BNF dose: Group 1, PBS 24 h before C. neoformans infection (control PBS); Group 2, BNF 0.5 mg Fe in 100 μl/animal 24 h before C. neoformans infection (BNF 0.5 mg); Group 3, BNF 0.05 mg Fe in 100 μl/animal 24 h before C. neoformans infection (BNF 0.05 mg); and Group 4, BNF 0.005 mg Fe in 100 μl/animal 24 h before C. neoformans infection (BNF 0.005 mg). Animals were observed daily for 14 days, surviving animals were euthanized, and tissues were extracted for fungal burden and histology analysis on 14 days after infection.

Experiment III: Study II results showed lower dose of BNF nanoparticles protected FVB/N mice from C. neoformans infection, motivating further study in the A/J strain with established susceptibility to C. neoformans infection. Thirty A/J mice (n = 10 animals per group) were divided into three groups: Group 1, PBS 24 h before C. neoformans infection (control PBS); Group 2, BNF 0.005 mg Fe in 100 μl/animal 24 h before C. neoformans infection (BNF 0.005 mg); and Group 3, BNF 0.0005 mg Fe in 100 μl/animal 24 h before C. neoformans infection (BNF 0.0005 mg). Five animals from each group were euthanized 14 days after infection for fungal burden, histology, and cytokine analysis, and the other five were followed for survival until 60 days.

Experiment IV: Another cohort of 20 A/J mice (n = 10 animals per group) were divided into two groups: Group 1, PBS 24 h before C. neoformans infection (control PBS); and Group 2, BNF 0.0005 mg Fe in 100 μl/animal 24 h before C. neoformans infection (BNF 0.0005 mg). All animals were sacrificed on day 14 for fungal burden and cytokine analysis from lung and spleen.

All mice challenged with C. neoformans were intranasally infected with 1 × 10⁷ C. neoformans yeast per animal, in a total volume of 20 μl (10 μl in each nasal cavity of the mouse). This model produces a pulmonary infection that rapidly disseminates to brain and other organs (41). Mice were anesthetized with 60 μL xylazine/ketamine solution intraperitoneally (95 mg of ketamine and 5 mg of xylazine per kilogram of animal body weight) to perform intranasal infection.

The fungal burden in lungs was measured in surviving mice by counting CFU (41, 42). At the endpoint of each experiment (as described above), mice were euthanized and the lungs (left lobe) were removed. Organ sections were weighed and homogenized in 1 mL of PBS. After serial dilutions, homogenates were inoculated on Sabouraud agar plates with 10 U/mL of streptomycin/penicillin. The plates were incubated at room temperature, and colonies counted after 48–72 h.

A piece of the right lung and a piece of the spleen were fixed in 10% formalin for 48 h before embedding in paraffin. Tissue slides were stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) for C. neoformans, and Prussian blue to visualize nanoparticles. The remaining right lungs and spleens of each A/J mouse treated with PBS or BNF 0.0005 mg were macerated with protease inhibitor (complete, EDTA-free, Roche Life Science, IN, USA) and centrifuged; supernatants of these samples were used for cytokine detection by sandwich-ELISA with commercial kits (BD OptEIA, BD Franklin Lakes, NJ, USA) for the following cytokines: IL-2 (#555148), IL- 4 (#555232), IL-10 (#555252), IFN-γ (#551866), and TNF-α (#555268). The protocol was followed according to the manufacturer's recommendations. Readings were performed in a plate spectrophotometer at 450 and 570 nm.

Immunofluorescence (IF) staining was performed on lung tissues to analyze the capsular polysaccharide covering Cryptococcus yeasts. Briefly, slides were deparaffinized on a slide warmer at 58°C 10 min followed by serial washing in 2 changes of xylene and in 100%, 95%, and 70% alcohol. Slides were dehydrated in deionized water and then treated with the antibody in a blocking solution for 30 min. The slides were then stained with the antibody (18B7) (43) conjugated with Oregon green during 16 h at 4°C. On the next day, all slides were washed with at least 5 changes of PBS and mounted with DAPI (4′,6-diamidino-2-phenylindole) containing mounting media (ProLong Gold Antifade Mountant, ThermoFisher Scientific, MA, USA). Slides were then visualized and imaged with a Zeiss microscope with ×100 magnification.

Statistical analyses were done using GraphPad Prism version 8.00 for Mac OS X (GraphPad Software, San Diego, CA, USA). One-way analysis of variance using a Kruskal-Wallis nonparametric test was used to compare the differences between groups, and individual comparisons of groups were performed using a Bonferroni posttest. The Student's t test was used to compare the number of CFU for different groups. The 90–95% confidence interval was determined in all experiments. Unpaired t test (F test to compare variances) was performed to compare cytokines data.