ABSTRACT
The prevalence of methamphetamine (METH) use is estimated at ∼35 million people worldwide, with over 10 million users in the United States. Chronic METH abuse and dependence predispose the users to participate in risky behaviors that may result in the acquisition of HIV and AIDS-related infections. Cryptococcus neoformans is an encapsulated fungus that causes cryptococcosis, an opportunistic infection that has recently been associated with drug users. METH enhances C. neoformans pulmonary infection, facilitating its dissemination and penetration into the central nervous system in mice. C. neoformans is a facultative intracellular microorganism and an excellent model to study host-pathogen interactions. METH compromises phagocyte effector functions, which might have deleterious consequences on infection control. In this study, we investigated the role of METH in phagocytosis and antigen processing by J774.16 macrophage- and NR-9460 microglia-like cells in the presence of a specific IgG1 to C. neoformans capsular polysaccharide. METH inhibits antibody-mediated phagocytosis of cryptococci by macrophages and microglia, likely due to reduced expression of membrane-bound Fcγ receptors. METH interferes with phagocytic cells’ phagosomal maturation, resulting in impaired fungal control. Phagocytic cell reduction in nitric oxide production during interactions with cryptococci was associated with decreased levels of tumor necrosis factor alpha (TNF-α) and lowered expression of Fcγ receptors. Importantly, pharmacological levels of METH in human blood and organs are cytotoxic to ∼20% of the phagocytes. Our findings suggest that METH abrogates immune cellular and molecular functions and may be deadly to phagocytic cells, which may result in increased susceptibility of users to acquire infectious diseases.
INTRODUCTION
Methamphetamine (METH) abuse is a serious public health issue in the United States and worldwide (1). METH is a potent and highly addictive stimulant to the central nervous system (CNS) that results in alterations of the users’ behavior, making them susceptible to engaging in unsafe activities like unprotected sex and sharing of contaminated needles, leading to the acquisition of infectious diseases (2). METH contributes to increased transmission of AIDS (2), hepatitis (3), tuberculosis (4), herpes (5), and other communicable diseases (6). Similarly, METH intake facilitates the acquisition of AIDS-associated infections. Cryptococcus neoformans is an encapsulated fungus that causes cryptococcosis, an opportunistic infection primarily in HIV-infected patients (7). Globally, this eukaryotic microorganism is responsible for approximately 223,000 cases of life-threatening meningoencephalitis and 181,000 deaths per year (8). Interestingly, recent cases in the United States of systemic cryptococcosis in intravenous drug users and a daily cannabis smoker suggest that drug abuse may exacerbate the disease even in the absence of HIV infection (9, 10). In this regard, METH enhances C. neoformans infection of the respiratory system and dissemination to the CNS of rodents by promoting fungal attachment, alteration of the polysaccharide capsule composition, release of immunosuppressive capsular material, and biofilm formation (11, 12). Thus, C. neoformans is an excellent model organism to answer questions regarding host-pathogen interactions in the setting of METH due to the accessibility to specific antibodies (Abs), cell lines, and animal models (13).
At pharmacological concentrations, METH exerts immunosuppressive effects on dendritic cells (14), neutrophils (15), and macrophages (16). Particularly, macrophages are important in controlling and containing C. neoformans infection in the lungs (17). Fcγ receptors (FcγRs) on macrophages can bind and mediate phagocytosis of Ab-opsonized yeast cells (18). Abs to the glucuronoxylomannan (GXM), the main component of the capsular polysaccharide, can modulate the infection (19). For instance, interaction of IgG1 complexes with related FcγRs facilitates either fungal killing, fungal growth inhibition through macrophage-mediated Ab-dependent cytotoxicity, macrophage phagocytosis, or neutrophil activation (20). In fact, passive capsule binding IgG1 therapy has been efficacious in inducing protective immunity, enhancing antifungal effectiveness, and prolonging survival in murine models of C. neoformans infection (19, 21).
C. neoformans is a facultative intracellular pathogen that resides in acidic phagosomes within macrophages (22). Cryptococci easily replicate and release abundant amounts of polysaccharide-enclosed vesicles inside phagocytic cells that accumulate in their phagosome, resulting in the escape of yeast cells through lytic and nonlytic exocytosis (23–25). Even though METH compromises the ability of macrophages to maintain acidic phagolysosomes (13, 16), the impact of this drug of abuse on the intracellular effects of specific Abs on the fate of a microbe within murine macrophages has not been extensively investigated. The intimate interaction of C. neoformans with macrophages is an ideal system to examine the role of METH in Ab function (13). Similarly and particularly important to cryptococcal infection, positron emission tomography has demonstrated that the highest accumulation and slowest clearance of METH in humans occur in the lungs and brain, respectively, with these organs being main disease-related targets of the fungus (26). In the brain, microglia, the resident surveillance cells of the CNS, act as its primary active immune defense and are associated with C. neoformans (27), suggesting that they play an important role controlling the infection (27, 28). In addition, microglia have been associated with METH-induced neurotoxicity (29, 30). Although microglia are vital in controlling microbial brain tissue colonization (27), their interactions with C. neoformans remain understudied. In this study, we explored the impact of METH on C. neoformans Ab-mediated phagocytosis and antigenic processing by J774.16 macrophage- and NR-9640 microglia-like cells. This study aimed to advance our understanding of how the innate immune system is affected at the molecular and cellular levels by METH abuse increasing the susceptibility of users to acquisition of infectious diseases.
RESULTS
METH inhibits IgG1-mediated phagocytosis of C. neoformans by J774.16 cells.We explored the impact of physiological METH concentrations on the phagocytosis of C. neoformans strain H99 by J774.16 macrophage-like cells in the presence of the capsule-specific IgG1 monoclonal Ab (MAb) 18B7 (Fig. 1). Light microscopy images of untreated J774.16 cells coincubated with MAb 18B7 demonstrated substantial phagocytosis and a minimal number of yeast cells present in their surroundings (Fig. 1A, left). Macrophages coincubated with 50 µM METH, MAb 18B7, and C. neoformans exhibited a considerable reduction in the uptake of the yeast cells (Fig. 1A, right). To corroborate the visual images, we measured the phagocytic indices of J776.14 cells treated with phosphate-buffered saline (PBS; untreated), 25 μM cytochalasin D (CytD; phagocytosis inhibitor; positive control), or 25 or 50 μM METH and coincubated with MAb 18B7 and C. neoformans (Fig. 1B). METH-treated macrophages coincubated with MAb 18B7-opsonized cryptococci displayed significantly lower phagocytosis than the untreated (P < 0.0001) and CytD-treated (P < 0.01) controls (Fig. 1B). CytD significantly impaired fungal phagocytosis by J774.16 cells relative to that of PBS-treated leukocytes (P < 0.0001) (Fig. 1B). These results confirmed previously published observations (14).
Methamphetamine (METH) inhibits capsular specific IgG1-mediated phagocytosis of C. neoformans by J774.16 macrophage-like cells. (A) Light microscopy images of untreated and 50 µM METH-treated 105 J774.16 cells incubated in the presence of anti-polysaccharide capsule-specific monoclonal antibody (MAb) 18B7 (IgG1; 2 µg/ml)-opsonized C. neoformans (106 cells). Images on the left side show control activated macrophage-like cells with phagocytized yeast cells. Black arrowheads indicate C. neoformans cells inside macrophages. Images on the right side show considerable numbers of cryptococci (black arrows) outside METH-treated macrophages. Scale bar: 20 µm. (B) The phagocytic indices (ratio of number of intracellular yeast cells to the number of macrophages) were determined after 2 h of incubation of 105 J774.16 cells with IgG1-opsonized C. neoformans (106 cells) in the absence (untreated) or presence of cytochalasin D (CytD; 25 µM; an inhibitor of actin polymerization and phagocytosis) or METH. Bars represent the means from four wells (100 cells per well), and error bars show SDs. Symbols (* and #) indicate P value significance calculated using analysis of variance analysis (ANOVA) and adjusted by use of the Bonferroni correction. “*” (P < 0.0001) and “#” (P < 0.01) indicate significantly higher fungal phagocytosis than in macrophages from the untreated and CytD groups, respectively. The experiments were performed twice, with similar results obtained. (C) Phagocytosis of FITC-conjugated MAb 18B7-labeled C. neoformans by J774.16 cells was determined using flow cytometry. Representative plots of internalized FITC-labeled fungi by macrophage-like cells are shown. Each plot was generated after 10,000 events were analyzed. (D) Confocal microscopy images of untreated and CytD- and METH-treated 105 J774.16 cells incubated in the presence of 106 C. neoformans cells opsonized with MAb 18B7 conjugated to FITC. Representative orthographic images of FITC-labeled cryptococci (green) ingested by macrophage-like cells (red, LAMP-1 stained, anti-CD107c conjugated to Alexa Fluor 647) are presented. For each main image, a top view of the field is shown, whereas the right and bottom images represent a Z-stack reconstruction. Scale bar: 20 µm.
We analyzed and confirmed the effects of METH on MAb 18B7-opsonized C. neoformans phagocytosis by macrophage-like cells using flow cytometry (Fig. 1C). METH reduced phagocytosis of the fungus by J774.16 cells, compared to the untreated control cells (Fig. 1C). Our results showed 68.4 and 75.7% phagocytosis inhibition in cells treated with 25 and 50 µM METH, respectively, compared to control cells. Macrophages treated with 25 μM CytD evinced a 55.6% phagocytosis decrease relative to the untreated cells. To differentiate between C. neoformans adhesion to the surface of J774.16 cells and fungal internalization, we performed in-depth microscopic examinations using confocal microscopy (Fig. 1D). Orthogonal images showed considerable internalization of cryptococci by control macrophage-like cells. In contrast, CytD- and METH-treated leukocytes evinced high number of free yeast cells in their surroundings, adhesion of fungal cells to their cell surface, and reduced phagocytosis. These data demonstrate that METH impairs IgG1-mediated phagocytosis of the fungi by macrophage-like cells.
METH reduces the expression of Fcγ receptors in J774.16 macrophage-like cells.Given that METH interferes with the ability of J774.16 cells to engulf cryptococci and the importance of FcγRs in recognizing antigen bound to IgG1, we investigated the effect of the drug on the expression of FcγRs using Western blot analysis (Fig. 2A). J774.16 macrophage-like cells treated with METH evinced a substantial reduction in the expression of FcγR molecules (CD16, -32, and -64) after incubation with C. neoformans (Fig. 2A). Leukocytes treated with 50 µM METH demonstrated significant reduction of CD16, -32, and -64 compared to the values for the untreated (P < 0.001) and CytD-treated (P < 0.001) groups (Fig. 2B). Similarly, the expression of CD64 was significantly reduced on J774.16 cells treated with 50 µM METH relative to those incubated with 25 µM METH (P < 0.001). Macrophage-like cells exposed to 25 µM METH displayed lower expression of CD16, -32, and -64 than did the untreated (P < 0.001) and CytD-treated (P < 0.001) groups (Fig. 2B). In addition, the expression of all FcγRs in macrophages was significantly affected by CytD compared to untreated cells (P < 0.001). To validate the results obtained by Western blotting, untreated cells and J774.16 macrophage-like cells exposed to 25 or 50 µM METH or CytD were stimulated with 10 µg/ml of IgG1-opsonized capsular polysaccharide and the mean fluorescence intensity (MFI) of CD64 was analyzed by flow cytometry (Fig. 2C). CytD- or METH-treated macrophages exhibited a significant MFI reduction by CD64 molecules compared to control cells incubated with PBS (P < 0.05) (Fig. 2C and D). Taken together, our findings suggest that IgG1-mediated phagocytosis of C. neoformans by macrophage-like cells is impaired by altering the expression of membrane-bound FcγRs.
METH reduces the expression of Fcγ receptors on the surface of J774.16 macrophage-like cells during interaction with C. neoformans or capsular polysaccharide alone. (A) The expression of CD16 (FcγRIII), CD32 (FcγRII), and CD64 (FcγRI) in macrophages was determined by Western blot analysis. A density of 105 J774.16 cells were incubated with PBS (untreated), 25 µM CytD, or 25 or 50 µM METH for 2 h, followed by interaction with capsule-specific IgG1-opsonized cryptococci (106). GAPDH was used as a control. (B) The levels of expression of CD16, CD32, and CD64 were measured by determining the relative intensity ratios. Individual band intensities from the Western blot in panel A were quantified using ImageJ software. GAPDH was used to determine the relative intensity ratios shown in panel B. Symbols (*, #, and ϕ) indicate P value significance (P < 0.001) calculated using ANOVA and adjusted by use of the Bonferroni correction. “*,” “#,” and “ϕ” indicate significantly lower macrophage Fcγ receptor expression than in the untreated, 25 µM CytD-treated, and 25 µM METH-treated groups, respectively. (C) The mean fluorescence intensity (MFI) of CD64 molecules was analyzed by flow cytometry after incubation of untreated (royal blue), 25 µM CytD-treated (red), and 25 µM (light blue) or 50 µM (yellow) METH-treated J774.16 cells (105) with IgG1-opsonized capsular polysaccharide (10 µg/ml) for 1 h. (D) The relative MFI of CD64 was determined after incubation of macrophages in the presence of PBS (untreated), 25 µM CytD, or 25 or 50 µM METH. The asterisk indicates P value significance (P < 0.05) calculated using ANOVA and adjusted by use of the Bonferroni correction, in this case representing significantly lower relative MFI in CD64 molecules in macrophages treated with 25 µM CytD or 25 or 50 µM METH relative to that of untreated leukocytes. All the experiments were performed twice, with similar results obtained.
METH alters phagolysosomal fusion and killing of C. neoformans by J774.16 cells.Capsule-specific IgG1 enhances J774.16 cell phagocytosis of C. neoformans and results in rapid fungal killing (21). We explored whether METH prevented IgG1-mediated killing of C. neoformans in macrophage-like cells (Fig. 3). J774.16 cells incubated with chloroquine (Chlq) (25 µM), a diprotic weak base and well known for raising the endocytic and lysosomal pH (14, 16), and METH (50 µM) demonstrated enhanced intracellular proliferation of yeast cells compared to untreated macrophages (P < 0.0001) (Fig. 3A). Although macrophages exposed to 25 µM METH exhibited an increasing trend for fungal proliferation, this was not significant. To further characterize the level of phagosomal maturation, since this is an important process for the killing of phagocytized cryptococci, we examined the association of the lysosome-associated membrane glycoprotein LAMP-1 with phagosomes treated with METH containing C. neoformans yeast cells using fluorescence microscopy. This was achieved by the labeling of LAMP-1 with anti-CD107 conjugated to Alexa Fluor 647 and fungal cells with MAb 18B7 conjugated to fluorescein isothiocyanate (FITC) (Fig. 3B). Fluorescent staining of LAMP-1 in untreated J774.16 cells infected with C. neoformans opsonized with MAb 18B7 brightly colocalized with the labeled yeast cells (Fig. 3B, top row). This pattern was seen in 66% of the untreated J774.16 phagosomes containing cryptococcal cells labeled with MAb 18B7 (Fig. 3C). LAMP-1 colocalization was not observed in images of Chlq-treated phagocytes (Fig. 3B, middle row) even when this pattern was apparent in 41% of phagosomes enclosing encapsulated yeast cells. LAMP-1 was seen diffusely for most part in METH-treated leukocytes (Fig. 3B, bottom row) containing yeast cells opsonized with MAb 18B7. However, we also observed intense colocalization around two specific cryptococcal cells (white arrows) and an absence of colocalization in other three cells (yellow arrowheads) (Fig. 3B;, bottom merged image). Similarly, METH-treated macrophages showcased larger nuclei and cell body size and apparent accumulation of polysaccharide capsule vesicles in the cytoplasm (green dots) compared to untreated controls (Fig. 3B, bottom row). Significantly less LAMP-1 staining occurred in METH-treated (P < 0.0001) and Chlq-treated (P < 0.0001) J774.16 cells than in untreated macrophages (Fig. 3C). These results indicate that METH reduces macrophage-phagolysosome fusion and stimulates C. neoformans capsular production and release within phagosomes, preventing the killing of the fungal cells (24).
METH interferes with the killing of C. neoformans cells opsonized with capsule-specific IgG1 by J774.16 cells. Macrophages (105) were first allowed to phagocytize MAb 18B7-opsonized C. neoformans H99 cells (106) for 2 h. Each well containing interacting cells was gently washed to get rid of fungal cells that were not phagocytized, and leukocytes containing cryptococci were incubated with feeding medium supplemented with either PBS (untreated), Chlq (25 µM), or METH (25 or 50 µM) for 24 h. (A) For the killing assay, phagocytic cells were lysed and fungal cells in the supernatant were plated and CFU were counted. Bars represent the means from four wells (three CFU counts per well), and error bars indicate SDs. The plus sign indicates P value (P < 0.0001) significance calculated using ANOVA and adjusted by use of the Bonferroni correction, in this case representing significantly higher survival of phagocytized fungi within macrophages than in the untreated group. (B) Light microscopy images show untreated and METH-treated macrophages with engulfed cryptococci. Immunofluorescent images show phagolysosomal fusion by localization of LAMP-1 (red; anti-CD107c conjugated to Alexa Fluor 647) to phagosomes of untreated and treated J774.16 cells containing cryptococcal cells labeled with MAb 18B7-conjugated to FITC (green). Nuclei of macrophages were stained in blue with DAPI. White arrows and yellow arrowheads indicate specific and absence of LAMP-1 colocalization, respectively. Scale bar, 20 μm. (C) Percentage of colocalization of LAMP-1 with C. neoformans in phagosomes of J774.16 cells. Bars represent the means of 20 macrophage phagosomes with phagocytized yeasts per condition, and error bars indicate SDs. Asterisks indicate P value significance (P < 0.0001) calculated using ANOVA and adjusted by use of the Bonferroni correction, in this case representing significantly lower percentage of LAMP-1 colocalization than in macrophages from the untreated group. The experiments were performed twice, and similar results were obtained. (D and E) Nitric oxide (NO) (D) and TNF-α (E) production was quantified using the Griess method and ELISA, respectively. Bars represent the means from four wells (triplicates per well), and error bars indicate SDs. Symbols (* and #) indicate P value significance (P < 0.05) calculated using ANOVA and adjusted by use of the Bonferroni correction. “*” and “#” indicate significantly lower NO and TNF-α production, respectively, than for the PBS- and Chlq-treated groups, respectively. These experiments were performed twice, and similar results were obtained.
Nitric oxide (NO) is produced by activated macrophages having antimicrobial activity against many intracellular pathogens (31). We assessed the impact of METH on NO production by J774.16 cells after C. neoformans phagocytosis. NO levels were significantly reduced in the supernatants of METH-treated macrophage-like cells compared with those of untreated controls (P < 0.05) (Fig. 3D). There was no difference observed between the Chlq and METH groups. Given that tumor necrosis factor alpha (TNF-α) regulates the production of NO synthase in macrophages (32), we investigated the levels of this proinflammatory cytokine in the supernatants of untreated and Chlq- and METH-treated cells after incubation with C. neoformans. TNF-α production was considerably reduced in the supernatants of METH-treated macrophage-like cells compared with that of untreated controls (P < 0.05) (Fig. 3E). J774.16 cells incubated with 50 µM METH also exhibited lower levels of this cytokine than did Chlq-treated cells (P < 0.05). We found that the amount of TNF-α produced by J774.16 cells was directly proportional to the levels of NO under all the conditions (Fig. 3D and E). This result was confirmed by using anti-TNF-α antibodies (data not shown). These data indicate that in addition to the negative effect of METH on macrophages’ phagosomal maturation, increased cryptococcal viability is enhanced by reduced production of NO and TNF-α during fungal interaction with these leukocytes.
METH compromises IgG1-mediated uptake of latex beads by macrophage-like cells.We assessed the phagocytic ability of the macrophages by exposing them to IgG1-opsonized latex beads (Fig. 4). CytD- and METH-treated J774.16 cells coincubated with IgG1-opsonized latex beads exhibited uptake similar to that of and phagocytosis significantly lower than that of the untreated (P < 0.0001) controls (Fig. 4A). Flow cytometry analysis validated that METH-treated macrophages had decreased phagocytosis of the latex beads, relative to the untreated control leukocytes (Fig. 4B). Our findings demonstrated 25.4 and 73.3% reductions in bead internalization in macrophages treated with 25 and 50 µM METH, respectively, compared to that of control cells. Macrophages treated with 25 μM CytD evinced a 32.5% phagocytosis reduction compared to the untreated cells. Light (Fig. 4C, top row) and fluorescent (Fig. 4C, bottom row) images displayed a considerable number of phagocytized IgG1-opsonized latex beads by untreated J774.16 cells after coincubation for 2 h. CytD- and METH-treated leukocytes had reduced uptake of Ab-opsonized beads and a substantial number of these free circular objects in their surroundings. Interestingly and even though CytD inhibited macrophage-like cells phagocytosis of latex beads, there were no drastic changes in the morphology of these phagocytic cells compared to the control cells. Nevertheless, METH-treated macrophages presented a smaller cell body than either untreated or CytD-treated cells, and this was consistent with exposure to an increased concentration of the drug (50 µM > 25 µM). These results indicate that METH interferes with IgG1-mediated phagocytosis, causes morphological alterations, and may be cytotoxic to phagocytic cells.
METH decreases phagocytosis of opsonized beads with capsule-specific IgG1 by J774.16 macrophage-like cells. (A) The phagocytic indices (ratio of number of intracellular beads to the number of macrophages counted) were determined after 2 h of incubation of 105 J774.16 cells with IgG1-opsonized latex beads (106) in the absence (untreated) or presence of CytD or METH. Bars represent the means from three wells (100 cells per well), and error bars indicate SDs. Asterisks indicate P value significance calculated using ANOVA and adjusted by use of the Bonferroni correction, in this case representing significantly (P < 0.0001) higher fungal phagocytosis than in macrophages from the untreated group. The experiments were performed twice, with similar results obtained. (B) Phagocytosis of FITC-conjugated IgG1-labeled latex beads by J774.16 cells was analyzed using flow cytometry. Representative plots of internalized FITC-labeled beads by macrophage-like cells are displayed. Each plot was generated after 10,000 events were analyzed. (C) Light (top row) and fluorescent (bottom row) microscopy images of untreated and CytD- and METH-treated leukocytes (105) incubated in the presence of 106 latex beads opsonized with IgG1 conjugated to FITC. Representative images of free beads (yellow, FITC) and beads internalized by J774.16 cells (red, LAMP-1; blue, DAPI) are shown. Scale bar: 10 µm.
METH causes apoptosis of J774.16 macrophage-like cells.METH causes morphological alterations of macrophages during their interactions with C. neoformans and latex beads. Thus, we investigated whether METH facilitates phagocytic cell apoptosis after staining with annexin V-FITC (green) and propidium iodide (PI; red) using flow cytometry. Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeative to PI. We performed a dose-response by exposing J774.16 cells to increased concentrations (3.125 to 50 μM) of METH. Flow cytometry of phagocytic cells validated that untreated macrophages evinced 89.2% viable, 4.9% early apoptotic, 0.4% late apoptotic, and 5.6% dead cells (Fig. 5A). In contrast, leukocytes treated with 3.125, 6.25, and 12.5 µM METH displayed ∼76% to 81% viable, ∼7% early apoptotic, ∼1% late apoptotic, and ∼10% to 16% dead cells (Fig. 5B to D). Cells treated with 25 µM METH exhibited an increased toxicity and early apoptotic population (13.1%), with only 68.5% viable, 1.7% late apoptotic, and 16.6% dead cells in the population (Fig. 5E). Interestingly, macrophages treated with 50 µM METH displayed viability (73.2%), apoptosis (12.2%), and mortality (14.6%) rates similar to those shown by cells exposed to 25 µM METH. Our data suggest that the physiological doses of METH used in these experiments are cytotoxic to ∼20% of the population of phagocytic cells.
METH induces apoptosis of macrophage-like cells. J774.16 cells were treated with increasing concentrations (3.125 [B], 6.25 [C], 12.5 [D], 25 [E], and 50 [F] μM) of METH for 2 h and compared to untreated cells (A). Apoptotic cells were analyzed by flow cytometry after being stained with annexin V-FITC together with propidium iodide (PI); representative plots are presented. The percentages of viable (live), early apoptotic (E apopt.), late apoptotic (L apopt.), and dead cells are reported. This experiment was performed twice, and similar results were obtained.
METH impairs phagocytosis and killing of C. neoformans by NR-9460 microglia-like cells.METH causes microglial activation and neurotoxicity (29, 30). Microglia, the primary immune cells of the CNS, are associated with C. neoformans and its GXM in brain tissue (27) and may therefore play a key role in cerebral meningoencephalitis defense and pathogenesis (33, 34). In addition, METH promotes C. neoformans dissemination from the respiratory tract into the brain parenchyma (11, 12). Due to C. neoformans neurotropism, we investigated the effect of METH on IgG1-mediated phagocytosis and killing of the fungus by NR-9640 microglia-like cells (Fig. 6). We observed a significant reduction in the phagocytosis of C. neoformans by microglial cells treated with CytD or METH compared to that by untreated cells (P < 0.05). However, 50 µM METH significantly impaired fungal phagocytosis by NR-9640 cells relative to that of microglia treated with PBS, 25 µM CytD, or 25 µM METH (P < 0.05) (Fig. 6A). Flow cytometry analysis demonstrated that NR-9640 cells exposed to 25 µM CytD and 25 and 50 µM METH had 22.3, 53.9, and 65.3% reductions, respectively, in internalization of cryptococci compared to that of control microglia (Fig. 6B). Fluorescent microscopy was used to visualize the uptake of C. neoformans cells and phagosomal maturation by microglial cells (Fig. 6C). Untreated microglia exhibited substantial phagocytosis of MAb 18B7-opsonized yeast cells and colocalization of LAMP-1 (Fig. 6C, top merged image, white arrows). This trend was observed in 80% of the untreated NR-9640 phagosomes containing cryptococcal cells labeled with MAb 18B7 (Fig. 6D). CytD-treated microglia showed reduced fungal internalization and phagosomal maturation (52%) (Fig. 6C and D). METH-treated microglia evinced several cryptococci on their cell surface (Fig. 6C, yellow arrowheads), minimal fungal uptake, and reduced phagosomal maturation (25 µM, 25%; 50 µM, 18.6%). Interestingly, several C. neoformans cells interacting with microglia exposed to either CytD or METH exhibited a MAb 18B7 punctate binding pattern (Fig. 6C, red arrows), which has been associated with nonprotective Abs (35). Colocalization of LAMP-1 inside NR-9640 cells was significantly lower in METH-treated cells than in untreated (P < 0.0001) and CytD-treated (P < 0.0001) control cells (Fig. 6D). Furthermore, we investigated the ability of NR-9640 cells to kill phagocytized MAb 18B7-opsonized cryptococci after treatment with METH (Fig. 6E). Exposure to METH significantly reduced fungal killing by microglial cells compared to that by untreated (P < 0.0001) and Chlq-treated (P < 0.0001) cells. Likewise, Chlq-treated microglia displayed significantly lower cryptococcal killing than untreated microglial cells (P < 0.0001). Production of NO by METH-treated microglia-like cells was also significantly reduced compared with that by untreated and Chlq-treated cells (P < 0.05) (Fig. 6F). We did not observe differences between the untreated and Chlq-treated groups. Similar to the case with J774.16 cells, TNF-α production was substantially decreased in the supernatants of METH-treated microglia-like cells compared with that of untreated controls (P < 0.05) (Fig. 6G). NR-9640 cells incubated with 50 µM METH also exhibited reduced levels of TNF-α compared to those in cells treated with 25 µM METH or Chlq (P < 0.05). TNF-α levels produced by NR-9640 cells were directly proportional to the amount of NO under all the conditions (Fig. 6G). Our findings demonstrate that METH interferes with IgG1-mediated phagocytosis and killing of C. neoformans by microglia-like cells.
METH compromises capsule-specific IgG1-mediated phagocytosis and killing of C. neoformans by NR-9460 microglia-like cells. (A) The phagocytic indices (ratio of number of intracellular yeast cells to the number of microglia) were determined after 2 h of incubation of 105 NR-9460 cells with IgG1-opsonized C. neoformans cells (106) in the absence (untreated) or presence of CytD or METH. Bars represent the means from four wells (100 cells per well), and error bars indicate SDs. Symbols (*, #, and ϕ) indicate P value significance (P < 0.05) calculated using ANOVA and adjusted by use of the Bonferroni correction. “*,” “#,” and “ϕ” indicate significantly higher fungal phagocytosis than in microglia from the untreated, 25 μM CytD-treated, and 25 μM METH-treated groups, respectively. (B) Phagocytosis of FITC-conjugated MAb 18B7-labeled C. neoformans by NR-9460 cells was determined using flow cytometry. Representative plots of internalized FITC-labeled fungi by microglia-like cells are shown. Each plot was generated after 10,000 events were analyzed. (C) Fluorescent microscopy images of phagolysosomal fusion by localization of LAMP-1 (red) to phagosomes of untreated and treated NR-9460 cells containing cryptococcal cells labeled with MAb 18B7 conjugated to FITC (green). Nuclei of microglial cells were stained in blue with DAPI. White arrows and yellow arrowheads indicate specific and absence of LAMP-1 colocalization, respectively. Red arrows indicate yeast cells with punctate MAb binding pattern. Scale bar, 10 μm. (D) Percentage of colocalization of LAMP-1 with C. neoformans in phagosomes of NR-9640 cells. Bars represent the means from 20 macrophage phagosomes with phagocytized yeasts per condition, and error bars indicate SDs. Symbols (* and #) indicate P value significance (P < 0.0001) calculated using ANOVA and adjusted by use of the Bonferroni correction. “*” and “#” indicate significantly lower percentages of LAMP-1 colocalization than in macrophages from the untreated and CytD-treated groups, respectively. (E) For the killing assay, microglial cells were lysed and fungal cells in the supernatant were plated and CFU were counted. Bars represent the means from four wells (three CFU counts per well), and error bars indicate SDs. Symbols (+ and &) indicate P value (P < 0.0001) significance calculated using ANOVA and adjusted by use of the Bonferroni correction. “+” and “&” indicate significantly higher survival of phagocytized fungi within microglia than in the untreated and Chlq-treated groups, respectively. (F and G) NO (F) and TNF-α (G) production was measured. Bars represent the means from four wells (triplicates per well), and error bars indicate SDs. Symbols (*, #, and ϕ) indicate P value significance (P < 0.05) calculated using ANOVA and adjusted by use of the Bonferroni correction. “*,” “#,” and “ϕ” indicate significantly lower NO and TNF-α production, respectively, than in the PBS-, Chlq-, and 25 µM METH-treated groups, respectively. For panels A to G, all experiments were performed twice, and similar results were obtained.
DISCUSSION
METH alters Ab responses against fungi in mice (16). Abs to the C. neoformans polysaccharide capsule enhance the antifungal activity of murine macrophages (21) and can promote phagocytosis (36), activate complement (37), modulate inflammation (19), inhibit biofilm formation (38), and reduce the shedding of capsular polysaccharide (39). Defects in humoral immunity are associated with increased susceptibility to acquire cryptococcosis in humans (40). We utilized J774.16 macrophage- and NR-9640 microglia-like cells to investigate the impact of METH on IgG1-mediated phagocytosis of C. neoformans. METH compromises the ability of macrophages and microglia to phagocytize yeast cells after opsonization with capsule-binding IgG1. Fungal cells aggregated closely and outside METH-treated phagocytes, indicating the inability of these peripheral and brain-resident cells to take up the yeast cells.
METH modifies the C. neoformans polysaccharide capsule’s carbohydrate composition and surface charge and stimulates its release during infection, resulting in biofilm formation and survival of the fungus (11). It is conceivable that changes to the capsular carbohydrate composition alter the binding epitopes of Abs, reducing their efficacy in tagging and targeting the fungus for phagocytosis. In this regard, we observed the presence of cryptococcal cells displaying a capsular punctate or limited GXM-binding pattern with MAb 18B7 during their interactions with METH-treated NR-9460 cells, and this is associated with no protection (35). Also, C. neoformans strains have shown slight structural variations on the carbohydrate composition of the capsule, exhibiting differences in the location of Ab binding and Ab-mediated changes in cell diameter and compressibility of the capsular polysaccharide (41). Similarly, Ab binding influences gene expression changes on C. neoformans and directly modulates fungal metabolism (42), a phenomenon also observed in Streptococcus pneumoniae which might be generalized in encapsulated microbes (43). Alternatively, it is also possible that METH modifies the glycosylation sites on the Ab molecule, compromising its binding affinity for GXM and protective antifungal activity. Structural studies of Ab molecules revealed that carbohydrate chains function to stabilize the immunoglobulin Fc structure and the interaction between the Ab molecule and its receptor (44, 45). Deglycosylation of MAb 18B7 has negative consequences in the Ab pharmacokinetics, decreasing its half-life in serum (46). Therefore, assessing the effect of METH on Ab structure is an interesting area that requires future investigations.
FcγRs on the surface of phagocytic cells recognize and bind to the Fc region of Abs that are attached to invading microbes (47). Since their activity stimulates macrophages to control and eliminate C. neoformans through IgG-mediated phagocytosis (20), we hypothesized that METH would impair the expression and distribution of FcγRs in these leukocytes, reducing fungal intake. Using Western blot analysis, we found that METH significantly decreases the expression of FcγRs on J774.16 cells. We observed multiple fungal cells surrounding METH-treated J774.16 cells, indicating that cryptococci accumulate outside macrophages, attributable to a reduced number of FcγRs available to mediate engulfment of the fungi. This substance of abuse may inhibit the tyrosine phosphorylation of the γ subunit of the receptor, which is necessary for phagocytosis of the cryptococcal cells (20). In addition, METH might lower the production of extracellular sphingosine-1-phosphate, a lipid product of the enzyme sphingosine kinase 1 which is required for Ab-mediated phagocytosis of C. neoformans (48), in macrophages.
METH may interfere with the signaling and function of FcγR-related cytoskeletal elements necessary for fungal internalization and phagosomal maturation (49). Flow cytometry analysis demonstrated that macrophages incubated with METH and sensitized with IgG1-opsonized GXM evinced a substantial MFI reduction by FcγR CD64 molecules. For example, FcγRs have been shown to modulate the inhibitory activity of infliximab, an IgG1 specific for TNF-α and used as a therapeutic in inflammatory bowel disease (50). Moreover, GXM-induced immunosuppression is mediated by direct interaction with FcγRIIB (51), highlighting the importance of FcγRs in the regulation of immunity. Even though addition of MAb to GXM reverses GXM-induced immunosuppression by shifting recognition of the polysaccharide antigen from FcγRIIB to FcγRIIA, it is probable that METH upregulates the Fas ligand in macrophages and other antigen-presenting cells, thus promoting T cell apoptosis and inhibition of a protective cell-mediated immunity which is important for controlling cryptococcosis (52, 53). Despite CytD’s well-known inhibitory effect on the cell’s cytoskeleton actin polymerization and a previous study showing its inhibition of internalization via FcγRs in phagocytes (54), to our knowledge this is the first report showing that CytD lowers the expression of FcγRs in macrophages. This important observation must be considered in future studies focusing on antibody-mediated phagocytosis or FcγR function.
Since the interaction between the IgG Fc domain and the FcγR is dependent on Ab glycosylation (55), it is plausible that METH-mediated deglycosylation of MAb 18B7 may promote inefficient complement-independent phagocytosis (46). This mechanism is postulated to involve the occurrence of Ab-mediated architectural change in C. neoformans capsular polysaccharide, which may encourage the fungus to interact directly with the complement receptors (56). Recent evidence supporting this possibility showed that METH mediates structural changes to the capsular composition (11) and inhibits complement-mediated phagocytosis (13) of C. neoformans. Interestingly, specific IgM to GXM enhances complement-mediated phagocytosis of the fungus (13), suggesting that the pentameric structure of this isotype may prevent METH-induced capsular modifications and positively influence the interaction of C. neoformans with complement receptors in macrophages.
While MAbs to fungi have been shown to enhance macrophage intracellular effector functions (57), METH has been also shown to interfere with antigenic processing and presentation by phagocytic cells (14, 16, 58). We found that METH inhibited macrophage and microglial intracellular killing of C. neoformans. Microscopic images demonstrated that METH-exposed J774.16 cells containing cryptococci had a larger size and their cytoplasm filled with capsular polysaccharide vesicles. This aligned with our previous observations showing that METH promotes fungal replication and release of capsular polysaccharide (11). It is apparent that METH is cytotoxic and may facilitate the accumulation of yeast cells and vesicles containing GXM in the phagosome and causes progressive permeabilization of the compartment’s membrane, leakiness of the polysaccharide into the cytoplasm, and exocytosis of the cryptococcal cells from macrophages (24). It has been previously proposed that intracellularly resident C. neoformans cells may switch the type and organization of capsular polysaccharide (24), a phenomenon that can be exacerbated by exposure to METH (11), making difficult the recognition and elimination of the exocytosed yeast cells by other intact immune cells.
The colocalization of LAMP-1 with fungal cells opsonized with specific MAbs stimulates effective antigen presentation (57). METH reduced LAMP-1 colocalization in the phagolysosome of macrophages and microglia containing C. neoformans cells. This compromises the capacity of LAMP-1 to process fungus-derived protein antigens into peptides that can be recognized by major histocompatibility complex (MHC) class II molecules (59, 60). In turn, this could affect the efficacy of phagocytes in presenting the antigen and activating T cells, since antigen interaction with MHC class II molecules is necessary for control of this fungal disease. We have shown that METH suppresses T cell proliferation when exposed to fungal antigens (16, 61).
NO production is necessary for Ab efficacy against C. neoformans in mice (62). NO production by phagocytic cells diffuses into phagosomes, where it can react with the mitochondrial respiratory chain and block electron transport as well as ATP biosynthesis, inhibiting fungal intracellular replication and regulation of the compartment milieu (31). We have previously shown that METH exposure significantly alters NO generation by murine macrophages (16). This study shows that NO production is significantly decreased in METH-treated J774.16 and NR-9640 cells containing MAb 18B7-opsonized cryptococci. The levels of NO produced by METH-treated macrophages and microglia were consistent with the levels of TNF-α produced. We have demonstrated that TNF-α has a direct effect on the expression of inducible NO synthase (13), the enzyme that catalyzes the production of NO through oxidation of l-arginine on the cytosolic side of the phagosome membrane. Likewise, there is a direct relationship between superoxide production by and expression of FcγRs on myeloid cells isolated from HIV-infected patients after interaction with MAb 18B7-opsonized C. neoformans (63). METH-mediated reduction of FcγRs on phagocytes is likely associated with the inability of these cells to produce NO during interactions with this encapsulated fungus.
METH induced apoptosis and death in ∼20% of J774.16 cells, suggesting that the reduction in fungal phagocytosis and antigenic processing may be in part associated with these adverse effects. We have demonstrated that METH decreases the number of phagocytic cells in the blood of treated BALB/c mice, enabling microbial infection (15). In addition, recent evidence indicates that METH induces apoptosis of microglia by altering mitochondrial function and cellular respiration (29). Even though it seems very difficult to resolve a mechanism of METH-mediated reduced phagocytosis of C. neoformans by macrophages and microglia when exposure to this substance of abuse is toxic to cells, previous studies support this notion and have shown an association between a reduction in the expression of Fcγ receptors and induced apoptosis in phagocytic cells (64, 65). Since C. neoformans has a predilection for brain tissue and METH enables fungal invasion of the CNS (12), it is conceivable that this substance of abuse facilitates cerebral cryptococcosis by compromising microglial function, inducing microglial cell-related neurotoxicity and facilitating cryptococcoma formation, resulting in meningoencephalitis. Although assessing the effects of METH on the cellular mediators of apoptosis and death in macrophages/microglia is important and such effects need further elucidation, this was out of the scope of the current study.
In conclusion, METH alters the ability of a capsule binding IgG1 to stimulate the effector functions of macrophages and microglia and killing of C. neoformans. Our findings suggest the importance of investigating the impact of METH on the molecular and cellular immunity of users and their susceptibility to acquire infectious diseases. Future in vivo studies testing the efficacy of passive administration and macrophage/microglial cell function against infectious microbes in the setting of METH abuse are warranted to understand the translational potential of the reported results in this study.
MATERIALS AND METHODS
C. neoformans.C. neoformans strain H99 (serotype A; isolated and kindly provided by John Perfect at Duke University) was inoculated in Sabouraud dextrose broth (Becton, Dickinson [BD]) and incubated at 30°C for 24 h in a rotary shaker (Thermo Fisher) set at 150 rpm.
Rationale for METH doses used in these studies.Controlled studies indicated that a single 260-mg dose peaks at a level of 7.5 μM (66). Thus, a single dose of 260 mg would be expected to produce 7.5 to 28.8 μM blood METH levels. Intravenous drug users tend to self-administer METH in binges, and as the drug exhibits a half-life of 11.4 to 12 h, this can lead to higher drug levels (67, 68). Binge patterns of use in individuals have shown that the fourth administration of 260 mg during a single day produces blood levels of 17 μM and could reach 20 μM on the second day of such a binge (66). Thus, binge doses of 260 to 1,000 mg produce 17 to 80 μM blood METH levels and levels in the micromolar range of hundreds in organs, including the brain and the spleen (69). Therefore, we selected ∼25 to 50 μM METH to perform our experiments.
J774.16 macrophage- and NR-9640 microglia-like cell lines.J774.16 is a well-characterized murine macrophage-like cell line that is extensively used to study C. neoformans-macrophage interactions (24, 70). J774.16 cells were generously provided by Arturo Casadevall at Johns Hopkins Bloomberg School of Public Health, Baltimore, MD. The murine microglial cell line NR-9460 is derived using brain tissue from wild-type mice (BEI Resources, NIAID, NIH). These microglial cells were immortalized by infection with the ecotropic transforming replication-deficient retrovirus J2. Characterization based on immunofluorescence, stimulation assays, and flow cytometry demonstrated that the NR-9460 cell line retains its microglia-specific morphological, functional, and surface expression properties.
Phagocytosis assay.Macrophage- or microglia-like cells were incubated in 96-well microtiter plates (Corning) in the absence or presence of METH (25 or 50 μM; Sigma) for 2 h. Cytochalasin D (25 μM; Sigma) was used as a positive control because it potently inhibits actin polymerization and phagocytosis (13, 15). Monolayers of J774.16 or NR-9640 cells were washed thrice with PBS, and feeding medium (13) supplemented with gamma interferon (IFN-γ; 100 U/ml) and bacterial lipopolysaccharide (LPS; 1 µg/ml) was added, followed by the addition of preincubated cryptococci with MAb 18B7 (anti-cryptococcal GXM IgG1 generated and generously provided by Arturo Casadevall; 2 μg/ml) for 1 h, in a macrophage/microglia (105 cells):yeast/latex bead (Thermo Fisher) (106 cells) ratio of 1:10. The plates were incubated for 2 h at 37°C and 5% CO2 for phagocytosis. For microscopic determination of phagocytosis, the monolayer coculture was washed thrice with PBS to remove nonadherent cells, Giemsa stained, fixed with cold methanol, and viewed with light microscopy with use of an Axiovert 200 M inverted microscope (Carl Zeiss) at a magnification of ×40. Images were collected using an AxioCam MrC digital camera using Zen 2011 digital imaging software (Carl Zeiss). The phagocytic index was determined to be the number of internalized yeast cells/latex beads over the number of 100 macrophages/microglia per well. Internalized cells were differentiated from attached cells because macrophages/microglia with internalized cryptococci tend to grow their cell body in the direction in which the intracellular fungal cells are present. For flow cytometry, adhered cells with internalized cryptococci/latex beads opsonized with IgG1 conjugated to FITC were removed after treatment with prewarmed Cellstripper solution (Corning) for 5 min at 37°C and 5% CO2. Samples were processed (10,000 events per sample) on a BD Accuri C6 flow cytometer, and C. neoformans phagocytosis by macrophages/microglia was analyzed using FCS Express software.
Macrophage viability assay.We assessed whether METH induces apoptosis in J774.16 cells, impairing their viability, and antimicrobial function. Apoptotic macrophages were analyzed by flow cytometry using an annexin V-FITC and PI kit (BD). Monolayers of 105 macrophages in a 96-well plate were incubated with increasing concentrations (3.125 to 50 µM) of METH for 2 h at 37°C and 5% CO2. After the desired incubation time, the medium was removed, 100 μl of prewarmed Cellstripper solution was added for 5 min to each well, and the plate was incubated at 37°C and 5% CO2. Macrophages were gently removed from each well, transferred to a flow cytometry tube, washed twice with cold PBS, and suspended in binding buffer. Five microliters each of annexin V-FITC and PI was added to cells. Each suspension was gently vortexed and incubated for 15 min at room temperature (RT; 25°C) in the dark. Then 400 μl of binding buffer was added to each tube and samples were analyzed (10,000 events per sample) by flow cytometry within 1 h using a BD Accuri C6 flow cytometer. The percentage of cells positive for PI (red) and/or annexin V-FITC (green) was determined using the FCS Express software. Untreated J774.16 cells were used as a control. Each condition was prepared and tested in triplicate.
Western blot analysis.To further understand the impact of METH on phagocytosis of C. neoformans, we assessed the expression of Fcγ receptors (CD16 [FcγRIII], CD32 [FcγRII], and CD64 [FcγRI]) in J774.16 cells. Macrophage-like cells at a density of 105 were incubated with PBS (untreated), 25 µM CytD, or 25 or 50 µM METH for 2 h, followed by interaction with 106 capsule-specific IgG1-opsonized cryptococci. Western blot analysis was conducted using cytoplasmic extracts made with an NE-PER nuclear and cytoplasmic extraction kit (Thermo Fisher). The mixture was centrifuged at 10,000 × g for 10 min at 4°C, and the resulting protein content of the supernatant was determined using the Bradford method, employing a protein assay kit (Bio-Rad). Lysates were preserved in protease inhibitor cocktail (Roche) and stored at −20°C until use. The supernatant was added with a sample buffer containing 1.6% sodium dodecyl sulfate, 5% glycerol, 0.1 M dithiothreitol, 0.002% bromophenol blue, and 62.5 mM Tris-HCl (pH 6.8). The mixture was then heated to 100°C for 5 min. Fifty-five micrograms of protein was applied to each lane of a gradient gel (10%; Bio-Rad). After electrophoresis at a constant 130 V/gel, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad; 0.2 mm) and briefly stained with Ponceau S (Sigma) to verify efficient transfer of the protein. The PVDF membrane was incubated for 1 h at 37°C in a blocking solution containing 5% nonfat dry milk, 0.04 M Tris-HCl (pH 7.6), 0.8% NaCl, and 0.5% Tween 20, followed by an overnight incubation at 4°C with mouse monoclonal anti-CD16 (dilution, 1:1,000; BD), anti-CD32 (dilution, 1:1,000; BD), or anti-CD64 (dilution, 1:1,000; BD) antibody and with subsequent addition of peroxidase-linked anti-mouse secondary IgG antibody diluted (1:5,000; Southern Biotech) in the blocking solution. Protein bands were detected and quantified with a lumino image analyzer (GE Typhoon 8600) after staining with chemiluminescence detection reagents (Thermo Fisher). Quantitative measurements of individual band intensities in Western blot analyses for CD16, CD32, and CD64 were performed using ImageJ software (National Institutes of Health). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; dilution, 1:1,000; BD), a cytoplasmic housekeeping protein, was used as a loading control to determine the relative intensity ratio.
GXM purification.GXM was isolated from C. neoformans strain H99 by filtration and ultrafiltration (11). Briefly, cryptococcal cells were cultivated in defined minimal medium (15 mM glucose, 10 mM MgSO4 · 7H2O, 29.4 mM KH2PO4, 0.13 mM glycine, 3 μM thiamine HCl [pH 6.0]) for 3 days at 30°C with shaking and separated from culture supernatants by centrifugation. The supernatant fluids were collected and centrifuged again to remove smaller debris. The pellets were discarded, and the resulting supernatant was concentrated approximately 20-fold using an ultrafiltration cell (100 kDa) with stirring and ultrafiltration discs. A nitrogen stream was used as the pressure gas. After the supernatant was concentrated, a thick, translucent film was observed in close association with the ultrafiltration disc and was covered by a concentrated fluid phase. The fluid phase was discarded, and the viscous layer was collected with a cell scraper for storage at room temperature. Fractions passed through the 100-kDa filtration discs were filtered through 10-kDa membranes, resulting again in film formation. For polysaccharide quantification, a capture enzyme-linked immunosorbent assay (ELISA) (71), the carbazole reaction for hexuronic acid (72), and the method for hexose detection described by Dubois et al. (73) were used. Each preparation was tested for contamination with LPS using the limulus amoebocyte lysate assay.
Determination of the MFI.J774.16 cells at a density of 105/ml were incubated with METH, CytD, or PBS as described above, stimulated with 10 µg/ml of IgG1-opsonized capsular polysaccharide for 1 h, washed three times with PBS, and then stained with anti‐CD64‐phycoerythrin (PE) (dilution, 1:1,000; BD) or its IgG1 isotype control. Samples were processed (10,000 events per sample) on a BD Accuri C6 flow cytometer, and the MFI of CD64 molecules in macrophages was analyzed using FCS Express software.
Killing assay.Since METH interfered with fungal phagocytosis, 105 J774.16 macrophage- or microglia-like cells were grown in feeding medium supplemented with IFN-γ (100 U/ml) and LPS (1 µg/ml), followed by the addition of preincubated 106 cryptococci with MAb 18B7 (2 μg/ml) for 1 h. Cryptococcal phagocytosis by macrophages/microglia occurred in an incubator for 2 h at 37°C and 5% CO2. Each well containing interacting cells was gently washed with feeding medium three times to get rid of fungal cells that were not phagocytized. Given that we were interested in the effect of METH on fungal killing and processing by these phagocytes, we incubated cryptococcus-engulfed macrophages/microglia with feeding medium supplemented with either PBS (untreated), METH (25 or 50 µM), or Chlq (25 µM) for 24 h at 37°C and 5% CO2 (16, 74). Macrophage- or microglia-like cells were lysed by forcibly pulling the culture through a 27-gauge needle five to seven times. A volume of 100 µl of suspension containing cryptococci was aspirated from the wells and transferred to a microcentrifuge tube with 900 µl of PBS. For each well, serial dilutions were performed and plated in triplicate onto Sabouraud dextrose agar plates, which were incubated at 30°C for 48 h. Viable cryptococcal cells were quantified as CFU. Although it is plausible that noninternalized cryptococci could replicate for several generations in feeding medium during a 24-h period, wells for each condition were microscopically monitored after phagocytosis to reduce the possibility of obtaining confounding results.
Fluorescence microscopy.J774.16 or NR-9460 cells phagocytized preopsonized C. neoformans/latex beads with MAb 18B7/IgG1 conjugated to FITC (green fluorescence) in eight-chamber tissue culture glass slides coated with poly-l-lysine (BD) for 2 h at 37°C and 5% CO2. Each well containing interacting cells was gently washed thrice and incubated with feeding medium supplemented with either PBS, 25 µM METH, or CytD or Chlq for 24 h. LAMP-1 is a type I transmembrane glycoprotein that is localized in lysosomes and endosomes. After incubation, samples were washed three times with PBS, permeabilized in −20°C methanol for 10 s, and incubated in blocking buffer (SuperBlock blocking buffer containing 2% goat serum) for 1 h at 37°C. The cells were incubated with anti-CD107a conjugated to Alexa Fluor 647 (red), which binds to LAMP-1 (dilution 1:100; BD), in blocking solution for 1 h at 37°C. The samples were washed thrice with blocking buffer and then incubated with 4′,6-diamidino-2-phenylindole (DAPI) (blue) to stain nuclei for 1 h at 37°C. The slides were washed again three times with PBS, coverslips were affixed, and the samples were viewed with an Olympus AX41 microscope (Olympus) with fluorescent filters attached. Fluorescent images were recorded with an Olympus DP70 camera and processed with Olympus DPC software. Finally, phagolysosomal fusion was analyzed by colocalization of LAMP-1 to J774.16 macrophage- or microglia-like cell phagosomes (n = 20) containing cryptococci using the overlay tool of ImageJ software. Briefly, obtained images were merged, and then the overlapped areas of the images were measured. Merged compartments with >95% overlap were determined to be colocalized. To distinguish between fungal adhesion to the surface of phagocytic cells and internalization, phagocytosis was carried out as described above, and microscopic examinations were performed with confocal microscopy. Depth measurements across the width of the device were taken at regular intervals using an upright Leica TCS SP5 confocal laser scanning microscope (Leica). To determine fungal internalization, a series of horizontal (x-y) optical sections with a thickness of 1.175 μm were taken throughout the full length of the phagocytic cells using a 60× objective. Confocal images of green (FITC) and red (LAMP-1) fluorescence were recorded simultaneously by using a multichannel mode. Z-stack images and measurements were corrected by using Leica LASX software (Leica) in the deconvolution mode.
NO and TNF-α determinations.NO and TNF-α produced in the supernatants of 105 macrophages/microglia treated with PBS, 25 µM Chlq, and 25 or 50 µM METH were quantified 24 h after incubation with 106 C. neoformans cells using the Griess method (Cayman Chemical; detection limit, 1 µM) and ELISA (BD; detection limit, 15.6 pg/ml) kits, respectively, according to the manufacturer’s protocol. NO and TNF-α levels were monitored by measuring the optical density at 540 nm and 405 nm, respectively, using a microtiter plate reader (Bio-Tek).
Statistical analysis.All data were subjected to statistical analysis using Prism 7.0 (GraphPad). P values for multiple comparisons were calculated by analysis of variance (ANOVA) and were adjusted by use of the Bonferroni correction. P values of <0.05 were considered significant.
ACKNOWLEDGMENTS
R.L.R. and L.R.M. are supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under award number 1R15GM117501-01A1. L.R.M. is funded and has an appointment in the Infectious Diseases and Immunology cluster of the Border Biomedical Research Center (BBRC; National Institute on Minority Health and Health Disparities [NIMHHD] award number 2G12MD007592), UTEP’s Research Centers in Minority Institutions Program.
All authors contributed to the design of the experiments, analysis of the data, and writing of the manuscript.
L.A., H.H.L., V.V.E., R.L.R., and L.R.M. declare no conflict of interest.
FOOTNOTES
- Received 7 February 2018.
- Returned for modification 26 February 2018.
- Accepted 27 November 2018.
- Accepted manuscript posted online 3 December 2018.
- Copyright © 2019 American Society for Microbiology.
