Immune responses induced by heat killed Saccharomyces cerevisiae: a vaccine against fungal infection.

Heat-killed Saccharomyces cerevisiae (HKY) used as a vaccine protects mice against systemic aspergillosis and coccidioidomycosis. Little is known about the immune response induced by HKY vaccination, consequently our goal was to do an analysis of HKY-induced immune responses involved in protection. BALB/c mice were vaccinated subcutaneously 3 times with HKY, a protective reagent, and bronchoalveolar lavage fluid, spleen, lymph nodes, and serum collected 2-5 weeks later. Cultured spleen or lymph node cells were stimulated with HKY. Proliferation of HKY-stimulated spleen or lymph node cells was tested by Alamar Blue reduction and flow cytometry. Cytokines from lymphocyte supernatants and antibody to glycans in serum collected from HKY-vaccinated mice were measured by ELISA. The results show that HKY promoted spleen cell and lymph node cell proliferation from HKY-vaccinated mice but not from PBS-vaccinated control mice (all P<0.05). Cytokine measurement showed HKY significantly promoted IFNγ, IL-6 and IL-17A production by spleen cells and lymph node cells (all P<0.05 and P<0.01, respectively). Cytokine production by HKY-stimulated cells from PBS-vaccinated mice was lower than those from HKY-vaccinated (P<0.05). Cytokines in BAL from HKY-vaccinated were higher, 1.7-fold for IFNγ and 2.1-fold for TNFα, than in BAL from PBS-vaccinated. Flow cytometry of lymphocytes from HKY-vaccinated showed 52% of CD3(+) or 56% of CD8(+) cells exhibited cell division after stimulation with HKY, compared to non-stimulated controls (26 or 23%, respectively) or HKY-stimulated cells from PBS-vaccinated (31 or 34%). HKY also induced antibody against Saccharomyces glucan and mannan with titers 4- or 2-fold, respectively, above that in unvaccinated. Taken together, the results suggested that HKY vaccination induces significant and specific Th1 type cellular immune responses and antibodies to glucan and mannan.

Effect of 3M-003, an imidazoquinoline, on phagocyte candidacidal activity directly and via induction of peripheral blood mononuclear cell cytokines.

3M-003, like related imidazoquinoline immunomodulators, interacts with Toll-like receptor-7 (TLR-7) and TLR-8. TLRs are important in the defense against fungal pathogens. The effect of 3M-003 on killing of Candida was evaluated on mouse (BALB/c) effector cell lineages: monocytes, neutrophils, and macrophages. After direct application, 3M-003 (1-80 microg mL(-1)) enhanced (P<0.05-0.01) macrophage killing, comparable to killing by interferon-gamma-activated macrophages. 3M-003 did not directly enhance the candidacidal activity of monocytes or neutrophils. To test an effect mediated by leukocytes, BALB/c peripheral blood mononuclear cells (PBMC) were stimulated in vitro with 3M-003 to generate cytokine-containing supernatants. 3M-003 at 1 or 3 microM was optimal for the stimulation of PBMC to produce tumor necrosis factor-alpha and interleukin-12p40 in 24 h. For indirect tests, monolayers were treated with supernatants for 18 h, the supernatants were removed, and effector cells were tested; the supernatants enhanced (P<0.05-0.01) killing, in 2-4-h assays, by neutrophils from 42% to 73%, macrophages from 0% to 23%, and monocytes from 0% to 20%. 3M-003, presumably through TLRs, acts directly on macrophages to enhance fungal killing and stimulates PBMC to produce soluble factors that enhance killing by neutrophils, macrophages, and monocytes. 3M-003 could be a candidate for antifungal immunotherapy.

Collectins and fungal pathogens: roles of surfactant proteins and mannose binding lectin in host resistance.

Collectins, collagenous carbohydrate-binding proteins (C-type lectins), are recognized as important factors in non-specific innate immune responses to pathogens. By binding surface carbohydrate structures of pathogens, collectins modify the interaction between pathogens and the immune system. We review the structure of the lung surfactant proteins (SP) SP-A and SP-D, and the serum collectins, mannose binding lectins; the binding of these collectins to pathogen associated molecular patterns or ligands on pathogenic fungi; and the effect of collectin binding to opportunistic and primary fungal pathogens on the interaction with host defense cells, which can result in enhancement or inhibition of resistance. The result of collectin binding to opportunistic fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Pneumocystis) or primary fungal pathogens (Blastomyces dermatitidis, Coccidioides, Histoplasma capsulatum, Paracoccidioides brasiliensis) on interaction with host defense cells relative to complement fixation, phagocytosis, and stimulation of cytokine/chemokine production is reviewed. Increased understanding of these relationships in future will help understand fungal pathogenesis and host defenses against mycoses.

Evasion of innate immune responses: evidence for mannose binding lectin inhibition of tumor necrosis factor alpha production by macrophages in response to Blastomyces dermatitidis.

Serum factors, including mannose binding lectins (MBL), influence innate responses to microbes. Little is known about the effects of serum factors or MBL on the interaction of Blastomyces dermatitidis, a pulmonary fungal pathogen, with macrophages or on tumor necrosis factor alpha (TNF-alpha) production. Since macrophage production of TNF-alpha is an important innate immune response, we examined a mouse peritoneal macrophage (PM) cell line (RAW) and resident PM from CD-1 mice to study TNF-alpha production by PM stimulated with heat-killed (HK) or live B. dermatitidis yeast cells. Mouse serum and heat-inactivated mouse serum inhibited TNF-alpha production 94% when macrophages were stimulated by B. dermatitidis, whereas mouse immunoglobulin G (IgG) did not have this effect. HK B. dermatitidis incubated with serum and then washed also failed to stimulate significant TNF-alpha production by PM. By the sandwich immunofluorescent antibody (IFA) method with anti-mouse MBL (MBL-A or -C), we showed that serum MBL bound to B. dermatitidis. When serum was absorbed with HK B. dermatitidis or live B. dermatitidis, absorbed serum failed to significantly inhibit TNF-alpha production by RAW cells plus B. dermatitidis, and immunoblotting showed that absorbed serum was depleted of MBL-C. If serum was absorbed with live B. dermatitidis, unbound serum was eluted, and bound serum factor(s) (BS) was released with guanidine buffer, BS inhibited TNF-alpha production by PM plus B. dermatitidis in a concentration-dependent manner. BS contained MBL-C, which bound B. dermatitidis, as shown by IFA assay. 1,3-beta-Glucan stimulated TNF-alpha production by PM, and this was inhibited by mouse serum. Treatment of B. dermatitidis with anti-1,3-beta-glucan antibody inhibited TNF-alpha production by PM. With anti-1,3-beta-glucan antibody, we showed by IFA assay that B. dermatitidis contained 1,3-beta-glucan. In an IFA study with B. dermatitidis, serum with an anti-mouse IgG conjugate did not result in fluorescence, yet serum blocked IFA staining of B. dermatitidis by anti-1,3-beta-glucan IgG antibody. This indicated that non-IgG serum factors binding to B. dermatitidis prevented access to 1,3-beta-glucan by anti-1,3-beta-glucan antibody. These results suggest that the mechanism of inhibition of the innate proinflammatory immune response of PM to B. dermatitidis is mediated by serum MBL binding to B. dermatitidis at 1,3-beta-glucan sites or sterically masking 1,3-beta-glucan sites, thus preventing 1,3-beta-glucan stimulation of PM for TNF-alpha production.

Production of IL-6, in contrast to other cytokines and chemokines, in macrophage innate immune responses: effect of serum and fungal (Blastomyces) challenge.

Murine peritoneal macrophages, after adherence and establishment in culture in vitro in the presence of medium containing fetal bovine serum (FBS) for 20 h, then cultured for 20 h, produced several cytokines. If, in the second 20 h period, a fungus (heat-killed Blastomyces, HK-Bd) was introduced, a more complex pattern of cytokine (particularly TNF) and chemokine production ensued. The cytokine production, assayed by antibody array and also quantitation in supernatants, was depressed (particularly TNF) by the addition of mouse serum to these cultures, with the exception of IL-6. Macrophages could be cultured in the presence or absence of serum during the initial 20 h adherence and establishment period, enabling study of the effect of serum factors. In the absence of serum, with or without fungal stimulation, cytokine and chemokine production was more restricted, largely to TNF and IL-6. The addition of mouse serum [corrected] resulted in marked depression of TNF and enhancement of IL-6. The combination of HK-Bd and mouse serum resulted in more IL-6 production than either component alone. The enhancement of IL-6 by mouse serum was concentration-dependent and maximal at 8 h. The effects of fungus or serum on macrophage production of cytokines were similar in an outbred and an inbred mouse strain. The larger repertoire of cytokine production in the macrophages that had been cultured longer (20 h+20 h) in serum may be related to maturation of cell receptors. IL-6 production in vivo in response to fungal-serum complexes could affect pathogenesis by opposing the host defense modulation by proinflammatory cytokines or by modulating the destructive effects of inflammation on host tissues.

IL-12 induction of resistance to pulmonary blastomycosis.

BACKGROUND: Why severity varies in blastomycosis outbreaks remains unresolved. In experimental pulmonary blastomycosis, susceptibility varied in mouse strains. In susceptible BALB/c the response is Th-2, in immunized, resistance is associated with Th-1. Can susceptibility be redirected by IL-12? Methods, results: BALB/c bronchoalveolar and peritoneal macrophages (PM) were shown deficient in IL-12 production in response to IFN-gamma+LPS. High dose IL-12 (1 microg, subcutaneously) treatment of BALB/c infected intranasally with Blastomyces resulted in enhanced survival (P<0.008). Since IL-12 was poorly tolerated, a new protocol for infected mice, IL-12 0.1 or 0.3 microg, every other day, resulted in minimal toxicity; almost all treated mice survived (P<0.002 vs. controls). When lungs of surviving mice were cultured, the 0.1 microg regimen resulted in fewer (P<0.02) cfu. For weeks after treatment, in vitro IFN-gamma treatment enabled PM Blastomyces killing. After infection spleen cells from IL-12 treated mice produced 4-fold more IFN-gamma and 3-fold less IL-10 in response to Blastomyces. IL-10 abrogated activation of macrophages by IFN-gamma for enhanced Blastomyces killing. CONCLUSIONS: A proper IL-12 treatment protocol induces resistance (survival and decreased growth in lungs), low toxicity, macrophage responsiveness to IFN-gamma for killing Blastomyces, up-regulation of IFN-gamma and down-regulation of IL-10 production.

Effect of lung surfactant collectins on bronchoalveolar macrophage interaction with Blastomyces dermatitidis: inhibition of tumor necrosis factor alpha production by surfactant protein D.

Alveolar surfactant modulates the antimicrobial function of bronchoalveolar macrophages (BAM). Little is known about the effect of surfactant-associated proteins in bronchoalveolar lavage fluid (BALF) on the interaction of BAM and Blastomyces dermatitidis. We investigated BALF enhancement or inhibition of TNF-alpha production by BAM stimulated by B. dermatitidis. BAM from CD-1 mice were stimulated with B. dermatitidis without or with normal BALF, surfactant protein A-deficient (SP-A-/-) or surfactant protein D-deficient (SP-D-/-) BALF, or a mixture of SP-A-/- and SP-D-/- BALF. An enzyme-linked immunosorbent assay was used to measure tumor necrosis factor alpha (TNF-alpha) in culture supernatants. BALFs were standardized in protein concentration. BAM plus B. dermatitidis (BAM-B. dermatitidis) TNF-alpha production was inhibited > or = 47% by BALF or SP-A-/- BALF (at 290 or 580 microg of protein/ml, P < 0.05 to 0.01); in contrast, SP-D-/- BALF did not significantly inhibit TNF-alpha production. If SP-A-/- BALF was mixed in equal amounts with SP-D-/- BALF, TNF-alpha production by BAM-B. dermatitidis was inhibited (P < 0.01). Finally, pure SP-D added to SP-D-/- BALF inhibited TNF-alpha production by BAM-B. dermatitidis (P < 0.01). B. dermatitidis incubated with BALF and washed, plus BAM, stimulated 63% less production of TNF-alpha than did unwashed B. dermatitidis (P < 0.05). SP-D was detected by anti-SP-D antibody on BALF-treated unwashed B. dermatitidis in an immunofluorescence assay (IFA). The BALF depleted by a coating of B. dermatitidis lost the ability to inhibit TNF-alpha production (P < 0.05). 1,3-beta-Glucan was a good stimulator of BAM for TNF-alpha production and was detected on B. dermatitidis by IFA. beta-Glucan incubated with BALF inhibited the binding of SP-D in BALF to B. dermatitidis as demonstrated by IFA. Our data suggest that SP-D in BALF binds beta-glucan on B. dermatitidis, blocking BAM access to beta-glucan, thereby inhibiting TNF-alpha production. Thus, whereas BALF constituents commonly mediate antimicrobial activity, B. dermatitidis may utilize BALF constituents, such as SP-D, to blunt the host defensive reaction; this effect could reduce inflammation and tissue destruction but could also promote disease.

Susceptibility to pulmonary blastomycosis in young compared to adult mice: immune deficiencies in young mice.

The immunological basis for differences in resistance to pulmonary blastomycosis between young (3 to 4-week-old) and adult (7 to 8-week-old) CD-1 mice is unknown. We assessed whether there were differences in fungicidal activity of phagocytes and Th-1 lymphocyte cytokine production. The fungicidal activity of young bronchoalveolar macrophages (BAM) (20%) against Blastomyces dermatitidis (Bd) was comparable to killing by adult BAM (25%). However, IFN-gamma enhanced the killing by adult BAM (from 30 to 69%) to a greater extent than BAM from young animals (from 20 to 30%). Killing of Bd by young peritoneal macrophages (PM) (46%) and adult PM (42%) was similar, and the enhancement of cells of both by IFN-gamma was similar. TNFalpha production by young macrophages (BAM or PM), when cocultured with Bd for 18 h, was half of TNFalpha secreted by adult macrophages. We found that polymorphonuclear neutrophils (PMN) from young mice had deficient fungicidal activity against Bd (37%) compared with adult PMN (80%). Interferon-gamma (IFN-gamma) treatment increased PMN killing of Bd by PMN of young animals from 37 to 80%. In an assessment of innate responses, we found spleen cells from young mice produced three-fold less IFN-gamma and three-fold less IL-2 than adult spleen cells in response to 1 microg/ml concanavalin A (Con A). The young spleen cells also produced more NO, which we demonstrated reduced Con A-induced proliferation. These in vitro results demonstrate several immunological deficiencies in cells from young mice and these deficiencies correlate with susceptibility. In a pilot reconstitution experiment in pulmonary blastomycosis, treatment of infected young mice with IFN-gamma (18.5 x 10(3) U, s.c.) on days 0, 1, and 2 significantly increased survival.

Inhibitor kappaB and nuclear factor kappaB in granulocyte-macrophage colony-stimulating factor antagonism of dexamethasone suppression of the macrophage response to Aspergillus fumigatus conidia.

BACKGROUND: The dexamethasone (DEX) immunosuppressive effect on macrophage killing activity and cytokine production in response to Aspergillus fumigatus conidia is antagonized by granulocyte-macrophage colony-stimulating factor (GM-CSF). The molecular mechanism is unknown. We postulated that this antagonism is mediated by inhibitor kappaB (I kappaB) induction by DEX and is opposed by acceleration of I kappaB degradation by GM-CSF with or without conidia stimulation, with corresponding effects on translocation and activation of nuclear factor kappa B (NF-kappaB). METHODS: We studied 2 types of cells, resident peritoneal macrophages from CD-1 mice and the murine macrophage RAW264.7 cell line. Cells were unstimulated or stimulated with conidia and simultaneously treated with DEX, GM-CSF, or DEX plus GM-CSF, for 2-4 hours. I kappaB degradation and NF-kappaB activation were assessed by Western blot. RESULTS: Macrophages stimulated with conidia alone increased NF-kappaB translocation. DEX increased I kappaB levels in cytoplasm and blocked translocation of NF-kappaB to the nucleus in unstimulated and conidia-stimulated macrophages. Conversely, GM-CSF decreased I kappaB levels. GM-CSF reversed the effect of DEX on I kappaB levels. NF-kappaB levels were minimal in DEX-treated macrophage nuclear extracts, compared with those from GM-CSF-treated and GM-CSF plus DEX-treated macrophages. CONCLUSION: GM-CSF can reverse the DEX immunosuppressive effect by enhancing I kappaB degradation and promoting NF-kappaB translocation. This would allow macrophage production of proinflammatory cytokines, facilitating resistance to aspergillosis.

Immunological basis for susceptibility and resistance to pulmonary blastomycosis in mouse strains.

The immunological basis for a >10-fold resistance of outbred CD-1 mice compared to inbred BALB/c mice to pulmonary blastomycosis was investigated. Bronchoalveolar macrophages (BAM) from CD-1 mice killed yeast cells of Blastomyces dermatitidis (Bd) by 25% and this increased to 59% when activated by IFN-gamma. In contrast, BAM from BALB/c mice lacked significant killing (5%) of Bd but could be activated by IFN-gamma for enhanced killing (19%). Peritoneal macrophages (PM) from CD-1 mice had significant fungicidal activity for Bd (43%) and this increased to 63% with IFN-gamma treatment. By contrast, PM from BALB/c mice did not significantly kill Bd (14%) but were activated by IFN-gamma for significant killing (24%). Fungicidal activity of peripheral blood polymorphonuclear neutrophils (PMN) from CD-1 (87%) was greater than that of BALB/c (75%) (P<0.05). Macrophage inflammatory protein-1alpha (MIP-1alpha) production by BAM from BALB/c was significantly less than that from CD-1 in response to co-culture with Bd. IFN-gamma production by CD-1 spleen cells in response to concanavalin A (Con A, 1microg/ml) was 8-fold greater than that by BALB/c spleen cells. In contrast, BAM and PM from BALB/c mice in co-culture with Bd secreted several-fold more TNFalpha than BAM or PM from CD-1 mice. IL-2 production by BALB/c spleen cells in response to Con A was 3- to 4-fold greater than that by CD-1 spleen cells. Depressed IL-2 production by Con A stimulated CD-1 spleen cells correlated with depressed proliferative responses. Resistance of CD-1 mice to pulmonary blastomycosis correlates with enhanced fungicidal activity of BAM, PM, PMN, and IFN-gamma production by Con A stimulated spleen cells, compared to BALB/c mice. Consistent with the in vitro enhancement of effector cell function by IFN-gamma, in vivo therapy with IFN-gamma significantly (P<0.0001) improved survival of BALB/c mice with pulmonary blastomycosis.

Combined action of micafungin, a new echinocandin, and human phagocytes for antifungal activity against Aspergillus fumigatus.

Micafungin, a new echinocandin, inhibits fungal cell wall beta-glucan synthesis. We postulated micafungin and host phagocytic cells could act together in damaging fungi. Using the metabolic XTT assay, micafungin alone (0.01 and 0.10 microg/ml) inhibited Aspergillus fumigatus germlings by 48% and 61%, respectively. Polymorphonuclear neutrophils (PMNs) inhibited germlings by 53%. Micafungin at 0.01 or 0.10 microg/ml and PMNs resulted in additive inhibition, 82% and 99%, respectively. Monocyte-derived macrophage (MDM) monolayers inhibited germling growth by 66%; micafungin (0.01 or 0.10 microg/ml) alone inhibited by 32% and 42%, respectively. MDMs and micafungin (0.01 or 0.10 microg/ml) caused an additive inhibition of growth, 85% and 95%, respectively. Hyphae were generated by incubation of conidia for 24 h with or without micafungin. PMNs alone, added to hyphae, inhibited growth by 19% in the subsequent 20 h. Hyphae generated in the presence of micafungin (0.10 microg/ml) and subsequently cultured with micafungin for 24 h inhibited growth by 64%. PMNs plus micafungin resulted in 82% inhibition. Monocytes alone inhibited hyphal growth by only 5%. Hyphae produced in the presence of micafungin (0.01 microg/ml) and incubated again with micafungin for 24 h inhibited growth by 47%; combination with monocytes resulted in 62% inhibition. These data indicate that micafungin inhibits growth of tissue forms of A. fumigatus, and phagocytes and micafungin together have an additive effect. These findings support the thesis that the greater efficacy of micafungin in vivo compared with in vitro could be due to combined effect of phagocytic cells and micafungin.

Regulation by granulocyte-macrophage colony-stimulating factor and/or steroids given in vivo of proinflammatory cytokine and chemokine production by bronchoalveolar macrophages in response to Aspergillus conidia.

Production of the proinflammatory cytokines interleukin (IL)-1 alpha and tumor necrosis factor (TNF)-alpha and of the chemotactic chemokine macrophage inflammatory protein (MIP)-1 alpha by bronchoalveolar macrophages (BAMs) from mice in response to Aspergillus conidia was tested after in vivo administration of saline, dexamethasone, cortisone acetate, granulocyte-macrophage colony-stimulating factor (GM-CSF), or a combination. Dexamethasone suppressed production of IL-1 alpha, TNF-alpha, and MIP-1 alpha; GM-CSF reduced secretion slightly but antagonized dexamethasone suppression when the two were given in combination. Cortisone acetate gave results similar to dexamethasone, but cortisone acetate suppression of BAM responses lasted 7 days, > or = 4 days longer than dexamethasone suppression. The effect of GM-CSF on cortisone acetate suppression lasted at least 7 days. GM-CSF could promote resistance to conidia by maintaining proinflammatory responses.

Regulation of bronchoalveolar macrophage proinflammatory cytokine production by dexamethasone and granulocyte-macrophage colony-stimulating factor after stimulation by Aspergillus conidia or lipopolysaccharide.

There is substantial evidence that local production of proinflammatory cytokines are very important in host resistance to aspergillosis. Dexamethasone (DEX) down-regulates production of these cytokines by stimulated bronchoalveolar macrophages (BAM) and constitutes a risk factor for aspergillosis. Granulocyte-macrophage colony-stimulating factor (GM-CSF) antagonizes DEX suppression of antifungal activity by BAM. Here we investigated the possibility that GM-CSF could antagonize DEX down-regulation of interleukin (IL)-1alpha and tumour necrosis factor (TNF)-alpha production by stimulated BAM. Control BAM responded to increasing numbers of conidia of Aspergillus fumigatus with increasing production of IL-1 and TNF. DEX (10(-7)M) significantly suppressed IL-1 and TNF production by BAM+conidia. Although GM-CSF did not enhance IL-1 or TNF production by BAM+conidia, GM-CSF significantly antagonized DEX suppression of IL-1 cytokine production. For comparative purposes, lipopolysaccharide (LPS, 1 microg/ml) was used to stimulate BAM in experiments similar to the above. In contrast to the findings with conidia, GM-CSF enhanced the production of IL-1 (5-fold) and TNF (1.5-fold) by LPS treated BAM. DEX suppression of cytokine production by BAM+LPS was modestly but significantly antagonized by GM-CSF. Moreover, differences between regulation of IL-1 and TNF production by BAM+conidia or LPS and peritoneal macrophages (PM)+conidia or LPS were documented. Finally, the anti-inflammatory cytokine IL-10 was minimally produced by BAM + conidia or LPS, but IL-10 was produced by PM + conidia or LPS. In summary, these data indicate that the risk factor for aspergillosis associated with DEX could be lessened in the pulmonary compartment with GM-CSF. On the other hand, desired effects of DEX could be maintained in other compartments.

Protection of peritoneal macrophages by granulocyte/macrophage colony-stimulating factor (GM-CSF) against dexamethasone suppression of killing of Aspergillus, and the effect of human GM-CSF.

Murine peritoneal macrophages in vitro could kill Aspergillus fumigatus conidia, and this activity could be suppressed with dexamethasone. Treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) alone did not boost killing, but GM-CSF treatment concurrently with dexamethasone reversed the dexamethasone suppression. Both recombinant human and recombinant murine GM-CSF were equivalent in this activity, even though the human reagent reportedly does not stimulate differentiation of murine stem cells. Recombinant human GM-CSF could also reverse dexamethasone suppression of bronchoalveolar macrophage conidiacidal activity. Sequential studies with peritoneal macrophages indicated that recombinant human GM-CSF pretreatment also blocked dexamethasone suppression, but the GM-CSF treatment given after dexamethasone did not block the suppressive effect. Recombinant human GM-CSF did not boost spleen cell proliferation to a mitogenic stimulus, and did not reverse dexamethasone suppression of proliferation. These studies suggest GM-CSF treatment prior to and concurrent with steroid immunosuppression may ameliorate the steroid effect on tissue macrophage antifungal activity, but does not affect steroid suppression of T-cell immunity.