Candidacidal Capacity Research Articles

Delinking CARD9 and IL-17: CARD9 Protects against Candida tropicalis Infection through a TNF-α-Dependent, IL-17-Independent Mechanism.

Candida is the third most common cause of bloodstream infections in hospitalized patients. Immunity to C. albicans, the most frequent species to be isolated in candidiasis, involves a well-characterized Dectin-1/caspase-associated recruitment domain adaptor 9 (CARD9)/IL-17 signaling axis. Infections caused by non-albicans Candida species are on the rise, but surprisingly little is known about immunity to these pathogens. In this study, we evaluated a systemic infection model of C. tropicalis, a clinically relevant, but poorly understood, non-albicans Candida. Mice lacking CARD9 were profoundly susceptible to C. tropicalis, displaying elevated fungal burdens in visceral organs and increased mortality compared with wild-type (WT) controls. Unlike C. albicans, IL-17 responses were induced normally in CARD9−/− mice following C. tropicalis infection. Moreover, there was no difference in susceptibility to C. tropicalis infection between WT and IL-23p19−/−, IL-17RA−/−, or Act1−/− mice. However, TNF-α expression was markedly impaired in CARD9−/− mice. Consistently, WT mice depleted of TNF-α were more susceptible to C. tropicalis, and CARD9-deficient neutrophils and monocytes failed to produce TNF-α following stimulation with C. tropicalis Ags. Both neutrophils and monocytes were necessary for defense against C. tropicalis, because their depletion in WT mice enhanced susceptibility to C. tropicalis. Disease in CARD9−/− mice was not due to defective neutrophil or monocyte recruitment to infected kidneys. However, TNF-α treatment of neutrophils in vitro enhanced their ability to kill C. tropicalis. Thus, protection against systemic C. tropicalis infection requires CARD9 and TNF-α, but not IL-17, signaling. Moreover, CARD9-dependent production of TNF-α enhances the candidacidal capacity of neutrophils, limiting fungal disease during disseminated C. tropicalis infection.

NADPH oxidase of human dendritic cells: Role in Candida albicans killing and regulation by interferons, dectin‐1 and CD206

Human monocyte-derived DC express the enzyme NADPH oxidase, responsible for ROS production. We show that Candida albicans did not activate NADPH oxidase in DC, and was poorly killed by these cells. However, Candida-killing activity increased upon DC stimulation with the NADPH oxidase activator PMA and was further enhanced by DC treatment with IFN-alpha or IFN-gamma. This fungicidal activity took place at high DC-to-Candida ratio, but decreased at low DC-to-yeast ratio, when Candida inhibited the NADPH oxidase by contrasting the assembly of the enzyme on DC plasma membrane. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated the PMA-dependent DC candidacidal capacity. Engagement of beta-glucan receptor dectin-1 induced NADPH oxidase activation in DC that was depressed by mannose-binding receptor CD206 co-stimulation. Candida was internalized by DC through mannose-binding receptors, but not through dectin-1, thus explaining why Candida did not elicit NADPH oxidase activity. Our results indicate that NADPH oxidase is involved in DC Candida-killing activity, which is increased by IFN. However, Candida escapes the oxidative damage by inhibiting NADPH oxidase and by entering DC through receptors not involved in NADPH oxidase activation.

Optimization of a myeloid cell transfusion strategy for infected neutropenic hosts

Although granulocyte transfusion is a logical, therapeutic option for neutropenic patients with refractory infections, significant technical barriers have prevented its widespread use. A novel phagocyte transfusion strategy has been developed based on activation of a human myeloid cell line HL-60. To further define the potential for HL-60 cells to recapitulate white cell transfusions, a shortened duration of activation was evaluated, facile quality control markers were defined, and the impact of low-dose irradiation on cell function was determined. Three days of activation resulted in increased cell viability and in vitro candidacidal capacity but with slightly higher cell replication compared with 7 days of activation. Cell viability and several flow cytometric measurements were accurate, quality control markers for HL-60 activation. In combination with activation, low-dose irradiation abrogated replication while sparing the candidacidal effects of the HL-60 cells. Infusion of irradiated, activated HL-60 cells improved survival of neutropenic, candidemic mice significantly. In summary, activated, irradiated HL-60 cells are microbicidal, have virtually no replicative capacity, and are safe and effective at protecting neutropenic mice against an otherwise 100% fatal candidal infection. With continued development, this strategy to recapitulate neutrophil functions has the potential to serve as an effective alternative to granulocyte transfusions.

Dialysis unmasks the fungicidal properties of glandular salivary secretions.

Several salivary proteins exhibit fungicidal activity against the opportunistic oral pathogen Candida albicans when they are tested as pure proteins in vitro. However, salivary secretions that are examined by the same assays either lack or exhibit very low candidacidal activity. Since ionic strength is known to have an inhibitory effect on the fungicidal activities of some proteins, parotid secretion was subjected to dialysis with membranes having molecular weight cutoffs (MWCOs) of 500, 1000, 10000, and 25000. Dialysis with membranes with MWCOs of >=1000 promoted fungicidal activity of parotid secretion, and this activity was dose dependent. The addition of sodium chloride to dialyzed, fungicidal parotid secretion abolished this activity, indicating that the fungicidal component was salt sensitive. Similar results were obtained with submandibular and sublingual secretions. Polyacrylamide gel electrophoresis under native and denaturing conditions was used to analyze the composition of the dialysate. Unexpectedly, proteins with MWs much lower than the nominal MWCOs of the membranes were not lost during dialysis. Among the retained proteins, the two fractions with MWs of approximately 17000 and 4000 exhibited fungicidal activity. These results are consistent with the presence of lysozyme and histatins, respectively, which may represent the major candidacidal capacity of dialyzed parotid secretion.

Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells.

Toll-like receptor (TLR) 2 and TLR4 play a pivotal role in recognition of Candida albicans. We demonstrate that TLR2(-/-) mice are more resistant to disseminated Candida infection, and this is associated with increased chemotaxis and enhanced candidacidal capacity of TLR2(-/-) macrophages. Although production of the proinflammatory cytokines TNF, IL-1alpha, and IL-1beta is normal, IL-10 release is severely impaired in the TLR2(-/-) mice. This is accompanied by a 50% decrease in the CD4+CD25+ regulatory T (Treg) cell population in TLR2(-/-) mice. In vitro studies confirmed that enhanced survival of Treg cells was induced by TLR2 agonists. The deleterious role of Treg cells on the innate immune response during disseminated candidiasis was underscored by the improved resistance to this infection after depletion of Treg cells. In conclusion, C. albicans induces immunosuppression through TLR2-derived signals that mediate increased IL-10 production and survival of Treg cells. This represents a novel mechanism in the pathogenesis of fungal infections.

Melanoma inhibits dendritic cell induction of cell-mediated immunity

Cell-mediated immunity is essential for tumor defense. Mature dendritic cells promote cell-mediated immunity through induction of Th1 responses from naı̈ve CD4+ T cells. This requires dendritic cell production of IL-12. Th1 responses facilitate generation of cytotoxic T cells from CD8+ precursors. Melanoma inhibits peritoneal macrophage function in vivo and in vitro. Peritoneal macrophages harvested from melanoma-bearing mice are less cytotoxic to B16 melanoma cells, have less candidacidal capacity, and produce less TNF-α, nitric oxide, and superoxide. Similar impairment of macrophage function has been demonstrated in vitro using B16 melanoma conditioned media. The purpose of this study was to determine whether melanoma inhibits dendritic cell maturation, IL-12 production, and/or subsequent T cell activation. Methods: Bone marrow-derived dendritic cells (DCs) were cultured from C57BL/6 mice using media containing GM-CSF and IL-4. Melanoma conditioned media (MCM) was generated by incubating B16 melanoma cells in control media for 24 hours. DCs were exposed to control or MCM for 24 hours prior to maturation with LPS (100 ng/ml). DCs and supernatants were harvested 24 hours after stimulation. DC maturation was measured by flow cytometry using antibodies to CD40, CD54, CD80, CD86, MHC class I, and MHC class II. IL-12 levels were measured by ELISA. DCs were pulsed with antigen, fixed, and co-cultured with T cells in the presence of control or MCM. IL-2 levels (a marker of T cell activation) were measured by ELISA from 24-hour supernatants. Results: Dendritic cells exposed to MCM produce less IL-12 in response to LPS than controls (82 pg/ml vs. 110 pg/ml, p = 0.0015). LPS induced DC maturation was not affected by exposure to MCM as surface expression of CD40, CD54, CD80, CD86, MHC class I, and MHC class II was similar. T cells activated by DCs in the presence of MCM produced 45% less IL-2 than controls (29 pg/ml vs. 53 pg/ml). Conclusion: Melanoma inhibits dendritic cell induction of cell-mediated immunity by decreasing IL-12 production and inhibiting T cell activation. However, dendritic cell maturation, as assessed by surface expression of classical markers, was not affected by MCM.

In vitro Effects of Clarithromycin on Human Polymorphonuclear Leukocyte Functions

Background: It has been shown that antibacterial agents affect polymorphonuclear leukocytes (PMNs), which are active in the body’s first line of defense, in different ways. However, few studies have investigated the effects of tablet dosage forms as opposed to pure powder forms. There is a need to demonstrate the clinical relevance of previous results with commercially available products. Methods: We examined the effects of clarithromycin solutions, prepared separately from either pure powder or commercially available tablets (250 mg), on human PMNs. The in vitro effects of each solution, adjusted to therapeutic concentration (1 mg/l), on PMN adherence, chemotaxis, phagocytosis, candidacidal capacity and superoxide production were studied. Results: Solutions prepared from pure clarithromycin powder did not affect the adherence, phagocytosis or superoxide production of PMNs, but did inhibit (p < 0.05) chemotaxis and candidacidal capacity. By contrast, a decrease (p < 0.05) in all functions except phagocytosis was observed with solutions prepared from the tablet dosage form of clarithromycin. Conclusion: The results of this study suggest that the tablet dosage form of clarithromycin may have a more pronounced inhibitory effect on human PMN functions than solutions prepared from the pure powder form.

Augmentation of human macrophage candidacidal capacity by recombinant human myeloperoxidase and granulocyte-macrophage colony-stimulating factor.

Phagocyte myeloperoxidase (MPO) is believed to be particularly important in defense against candida infection. We reported earlier that monocytes, rich in MPO, killed Candida albicans at a significantly higher rate and extent than did monocyte-derived macrophages, known to lack MPO, and that C. albicans is less resistant to MPO-dependent oxidants than less pathogenic Candida species. We hypothesized, therefore, that the capacity of macrophages to kill C. albicans might be improved in the presence of MPO. In this study, we evaluated the ability of recombinant human MPO (rhMPO) to augment the killing of C. albicans by resident macrophages and macrophages activated by recombinant human granulocyte-macrophage colony-stimulating factor. Addition of rhMPO (concentration range, 0.8 to 6.4 U/ml) to suspensions of resident and activated macrophages and opsonized C. albicans resulted in concentration-dependent and significant increases in candida killing. This enhancement was particularly pronounced with activated macrophages, whether C. albicans was opsonized or unopsonized and ingested through the macrophage mannose receptor. rhMPO did not affect the killing of C. albicans by monocytes, nor did it affect phagocytosis of opsonized or unopsonized C. albicans. These results indicate that exogenous rhMPO can augment the candidacidal capacity of both resident and activated macrophages, with a more profound effect on activated cells. We suggest that rhMPO may be effective in the treatment of invasive candidiasis.

INVASIVE CANDIDAL INFECTIONS: BASIC IMMUNE MECHANISMS OF HOST DEFENSE AND FUTURE PERSPECTIVES FOR THERAPY 6

Invasive candidosis poses a serious threat to immunocompromised patients. We studied the functional and biochemical basis of phagocytosis and killing of candida by human monocytes (Mo) and monocyte-derived macrophages (MDM). We found that opsonized candida species were ingested equally by Mo and MDM, but uptake of unopsonized candida was mediated only by MDM, primarily through the macrophage mannose receptor. Mo killed C.parapsilosis, rarely pathogenic, significantly better than C.albicans, a serious pathogen. In contrast to Mo, macrophages killed both species to an equivalent extent. The two species triggered the respiratory burst and release of myeloperoxidase (MPO) to an equivalent extent. C.parapsilosis was killed much more easily than C.albicans by exposure to low concentrations of hypochlorite or monochloramine, MPO-dependent oxidants released by Mo but not macrophages, which lack MPO. With six different candida strains there was a significant correlation between killing by Mo and susceptibility to hypochlorite (r=0.926) or monochloramine (r=0.981)(p<0.01 for each). Therefore, we hypothesized, that the capacity of macrophages to kill candida might be retained in the presence of MPO and studied the ability of rhMPO to augment the fungicidal activity of resident and rhGM-CSF-activated macrophages for C.albicans. Addition of rhMPO (concentrations: 0.8, 1.6, 3.2, and 6.4 U/ml) to suspensions of resident macrophages and serum-opsonized C.albicans resulted in significant increases in the killing of candida at concentrations of 3.2 U/ml and 6.4 U/ml(p<0.05 for each). Addition of rhMPO (concentrations: 0.8 to 6.4) to activated macrophages further increased the candidacidal capacity of rhGM-CSF-activated cells (p<0.05 at each concentrations used). These data indicate that exogenous rhMPO augments candidacidal capacity of both resident and rhGM-CSF-activated macrophages with a more profound effect on activated cells. These data suggest that rhMPO might be effective in the treatment of invasive candidiasis.

Nitric oxide enhances resistance of SCID mice to mucosal candidiasis.

The capacity of macrophages from SCID and C.B-17 mice to kill Candida albicans via a nitric oxide (NO)-dependent pathway and the contribution of NO in resistance to mucosal candidiasis were assessed. In vitro, an inhibitor of NO synthase (NOS) reduced the candidacidal activity and nitrite-producing capacity of activated resident peritoneal macrophages from immunocompetent C.B-17 and immunodeficient SCID mice. In vivo, stomachs from gnotobiotic SCID mice that were colonized with a pure culture of C. albicans had low-grade infections and expressed inducible NOS (iNOS) mRNA. C. albicans-monoassociated SCID mice treated with an inhibitor of NOS had more severe orogastric candidiasis than controls. These data suggest that NO contributes to the candidacidal capacity of activated macrophages from C.B-17 and SCID mice and that NO synthesized by iNOS may contribute to the resistance of SCID mice to mucosal candidiasis.

Effects of cefpirome in comparison with cefuroxime against human polymorphonuclear leucocytes in vitro

The interaction of cefpirome and cefuroxime, with human polymorphonuclear leucocytes (PMNs) was examined. The effects of therapeutic concentration (1, 10, and 50 mg/L) of both antimicrobial agents on the adherence, spontaneous mobility, chemotaxis, chemokinesis, phagocytosis and candidacidal capacity were studied in vitro. No statistically significant variations, in relation to the control, were observed in the ability of PMNs to adhere to nylon fibre at the concentrations of cefuroxime and cefpirome used. With both antimicrobial agents, PMN mobility increased as the antibiotic concentration was increased. A statistically significant increase (P < 0.05) in spontaneous mobility, chemotaxis and chemokinesis was observed with cefpirome at only 10 mg/L. However, only spontaneous mobility was increased significantly with cefuroxime at 10 mg/L. The strongest effects were observed with cefpirome at 50 mg/L with variations of about 70% (P < 0.01), in chemotaxis and chemokinesis. In general, cefpirome had a positive effect on phagocytosis and candidacidal power, which was significant at a concentration of 50 mg/L (P < 0.01). Cefuroxime, in general, produced no modifications in either phagocytosis or candidacidal power.

Activity of vancomycin and teicoplanin against human polymorphonuclear leucocytes: a comparative study

The in-vitro effects of therapeutic concentrations of teicoplanin and vancomycin on the adherence, spontaneous mobility, chemotaxis, phagocytic and candidacidal capacity of human polymorphonuclear leukocytes (PMNs) were studied. Adherence was not affected by the tested concentrations of teicoplanin. A progressive diminishing of adherence was observed in relation to the control, which reached 30% with the maximum concentration of vancomycin (50 mg/L), although this was not statistically significant. An increase in spontaneous mobility was noted after 30 min incubation in the presence of any one of the tested concentrations of both antibiotics, although the results were only significant (P less than 0.05) for 50 mg/L of teicoplanin. Chemotaxis increased, although not significantly (P greater than 0.05) in the presence of 10 and 50 mg/L of teicoplanin, and diminished with respect to the control very significantly (P less than 0.019) when 1 and 50 mg/L of vancomycin were tested. The study of the phagocytic and candidacidal capacity of the PMNs on Candida albicans showed that teicoplanin had a favourable effect on both functions, even though the differences were not statistically significant.

The effect of pentamidine salts on the NADPH-dependent oxidase of stimulated neutrophilic grannlocytes

Both pentamidine isethionate and pentamidine mesylate induced a depression in activity of the NADPH-dependent oxidase system of stimulated human neutrophilic granulocytes. This drug-induced effect occurred at concentrations of 0.7, 1.1 and 1.5 mg/l, values within the therapeutic range after parenteral administration of a standard dose of either pentamidine salt, and was dose-related. There was no significant difference between the two salts with regard to this suppression in neutrophilic granulocyte function. The reduced activity of the NADPH-dependent oxidase system, after incubation with pentamidine salts, may be associated with the previously observed depression in candidacidal capacity of human neutrophilic granulocytes treated with these drugs.

Augmentation of human blood monocyte microbicidal activity by RU 41740, a glycoprotein extract from Klebsiella pneumoniae.

Non-specific activation of host defences may have a significant impact on the outcome of infections in the immunocompromized patient. RU 41740, a glycoprotein extract from Klebsiella pneumoniae, effective in increasing resistance to experimental infections in animals, has been examined in vitro for its effect on human blood monocyte locomotion, phagocytosis, killing of Candida albicans, and release of superoxide anion. RU 41740 had no chemo-attractant activity nor any effect on monocyte chemotactic and phagocytic function. Candidacidal capacity and superoxide anion production by monocytes were significantly enhanced after preincubation with RU 41740 greater than 1.0 microgram/ml. The effect was dose- and time-dependent and was not influenced by the presence of lymphocytes or their culture supernatants. This suggests a direct interaction with monocytes as the mechanism of action of the extract.

Enhanced killing of Candida albicans by cultured peritoneal exudate cells treated with SM-1213, a synthetic immunomodulator.

The effect of SM-1213 [1,2-O-isopropylidene-3-O-3'-(N', N'-dimethylamino-n-propyl)-D-glucofuranose] on the candidastatic and candidacidal capacity of guinea pig peritoneal exudate cells was investigated. Phagocytes treated with SM-1213 demonstrated an enhanced ability to inhibit intracellular hyphal formation and elongation of Candida albicans cells as well as to reduce the percentage of viable cells remaining after phagocytosis. Significant drug-induced anticandidal effects were also observed when peritoneal exudate cells were preincubated with SM-1213 and washed before infection with C. albicans. SM-1213 had no direct anticandidal effect against the yeast or hyphal form of the fungus, suggesting that the observed candidastatic and candidacidal effect of this drug was due to the enhancement of antimicrobial functions of phagocytes.

Oral clotrimazole in the treatment of esophageal candidiasis

Several antifungal regimens had failed to relieve severe, recurrent esophageal candidiasis in a 75 year old woman without predisposing disease whose serum transiently inhibited the candidacidal capacity of her polymorphonuclear leukocytes. Treatment with oral nystatin suspension was unsuccessful, whereas intravenous amphotericin B and miconazole induced only transient responses. Oral 5-fluorocytosine induced severe nausea and vomiting, and was discontinued. Oral clotrimazole troches produced prompt and sustained eradication of the patient's candidal esophagitis.

A serum-dependent defect of neutrophil function in chronic mucocutaneous candidiasis.

Investigations into the immunological functions of three patients with chronic mucocutaneous candidiasis were carried out. Phagocytosis of Candida albicans, nitroblue tetrazolium dye reduction, complement, and immunoglobulin levels were normal. Candidacidal assays using the neutrophils of the patients in autologous serum showed significant decreases below normal levels. This decrease in kill could be corrected by incubating the patients' cells in normal control or human AB serum. Cross-over serum studies also showed that the patients' sera had inhibitory effects on the candidacidal capacity of normal neutrophils. These changes in kill were related to candida antibody levels.

Defective Candida killing in childhood malnutrition.

This study shows that malnourished children have impaired candidacidal activity of leucocytes, a finding which runs parallel to the higher rates of isolation of Candida sp. from throat secretions. In well nournished subjects the mean candidacidal ability was 44.5% in the moderately undernourished group it was 17.6%, and in the severely malnourished group it was 13.7%. This impaired candidacidal capacity may be important in the establishment of large numbers of Candida sp. which are commonly found in the upper intestine and in the pathogenesis of diarrhoea in children with malnutrition.

Recovery of functional capacity of human granulocytes after freeze-preservation with dimethyl sulphoxide

Huma peripheral blood leucocytes (neutrophil rich) were collected either with preservative-free heparin (PFH) or acid citrate dextrose (ACD), frozen with dimethyl sulphoxide (DMSO) at a controlled rate, stored in liquid nitrogen at −196 °C and reconstituted in a solution containing dextran polymer 70. A battery of tests including nitroblue tetrazolium (NBT) reduction, Candida phagocytic and candidacidal capacity was used to compare anticoagulants and reconstitution methods as they affect functional capacity of freeze-thawed neutrophils during a short post-thaw period. Heparin showed an overall advantage over acid citrate dextrose. A slow titration reconstitution method did not improve cell yield or functional capacity compared with a rapid dilution method and was a more cumbersome technique. The presence of complement greatly improved the capacity of reconstituted cells to reduct NBT and synthesize formazan. Freeze-thawed cells showed a selective response to stimulation as judged by the quantitative NBT test, responding strongly to zymosan in comparison with E. coli endotoxin. Lignocaine hydrochloride added to the reconstituent medium in concentrations up to 20 mmol/l did not have additional protective effect on post-thawed leucocytes as assessed by agglutination and leucocyte yields when compared with reconstitutent solutions containing only dextran. Reducing pH did not significantly slow the rate of gelling and leuco-agglutination or improve cell yields. Using these findings to optimize conditions reconstituted neutrophils retained 31.7% of fresh NBT activity, 27.6% of fresh phagocytic, and 22.3% of fresh candidacidal capacity.

PHAGOCYTOSIS AND CANDIDACIDAL ABILITY OF THE LEUCOCYTES IN THE NEWBORN; EFFECT OF EXCHANGE TRANSFUSION

The phagocytosis and killing capacity of candida albicans by the newborn's leucocytes were studied in order to investigate the increased incidence of monilia infection during the first days of life. They were both found to be deficient. The effect of exchange transfusion was also examined by studying the phagocytosis, candidacidal capacity and the C3 component of the compliment in the serum before, at the end and 72 hours following as well as in the donors blood. It was found that: 1) following ET phagocytosis tends to drop returning to the pretransfusion levels at 72 hours. 2) The exchange transfusion did not have any influence on the candidacidal activity of the leucocytes nor on the concentration of C3 component of the compliment.