Protection against experimental cryptococcosis elicited by Cationic Adjuvant Formulation 01-adjuvanted subunit vaccines.

The fungal infection, cryptococcosis, is responsible for >100,000 deaths annually. No licensed vaccines are available. We explored the efficacy and immune responses of subunit cryptococcal vaccines adjuvanted with Cationic Adjuvant Formulation 01 (CAF01). CAF01 promotes humoral and T helper (Th) 1 and Th17 immune responses and has been safely used in human vaccine trials. Four subcutaneous vaccines, each containing single recombinant Cryptococcus neoformans protein antigens, partially protected mice from experimental cryptococcosis. Protection increased, up to 100%, in mice that received bivalent and quadrivalent vaccine formulations. Vaccinated mice that received a pulmonary challenge with C. neoformans had an influx of leukocytes into the lung including robust numbers of polyfunctional CD4+ T cells which produced Interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL)-17 upon ex vivo antigenic stimulation. Cytokine-producing lung CD8+ T cells were also found, albeit in lesser numbers. A significant, durable IFNγ response was observed in the lungs, spleen, and blood. Moreover, IFNγ secretion following ex vivo stimulation directly correlated with fungal clearance in the lungs. Thus, we have developed multivalent cryptococcal vaccines which protect mice from experimental cryptococcosis using an adjuvant which has been safely tested in humans. These preclinical studies suggest a path towards human cryptococcal vaccine trials.

Dysregulated pulmonary inflammatory responses exacerbate the outcome of secondary aspergillosis following influenza.

IMPORTANCE: Severe influenza is a risk factor for fatal invasive pulmonary aspergillosis; however, the mechanistic basis for the lethality is unclear. Utilizing an influenza-associated pulmonary aspergillosis (IAPA) model, we found that mice infected with influenza A virus followed by Aspergillus fumigatus had 100% mortality when superinfected during the early stages of influenza but survived at later stages. While superinfected mice had dysregulated pulmonary inflammatory responses compared to controls, they had neither increased inflammation nor extensive fungal growth. Although influenza-infected mice had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus, influenza did not affect the ability of neutrophils to clear the fungi. Our data suggest that the lethality seen in our model of IAPA is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. If confirmed in humans, our findings provide a rationale for clinical studies of adjuvant anti-inflammatory agents in the treatment of IAPA.

Protection of Mice against Experimental Cryptococcosis by Synthesized Peptides Delivered in Glucan Particles.

The high global burden of cryptococcosis has made development of a protective vaccine a public health priority. We previously demonstrated that a vaccine composed of recombinant Cryptococcus neoformans chitin deacetylase 2 (Cda2) delivered in glucan particles (GPs) protects BALB/c and C57BL/6 mice from an otherwise lethal challenge with a highly virulent C. neoformans strain. An immunoinformatic analysis of Cda2 revealed a peptide sequence predicted to have strong binding to the major histocompatibility complex class II (MHC II) H2-IAd allele found in BALB/c mice. BALB/c mice vaccinated with GPs containing a 32-amino-acid peptide (Cda2-Pep1) that included this strong binding region were protected from cryptococcosis. Protection was lost with GP-based vaccines containing versions of recombinant Cda2 protein and Cda2-Pep1 with mutations predicted to greatly diminish MHC II binding. Cda2 has homology to the three other C. neoformans chitin deacetylases, Cda1, Cda3, and Fpd1, in the high-MHC II-binding region. GPs loaded with homologous peptides of Cda1, Cda3, and Fpd1 protected BALB/c mice from experimental cryptococcosis, albeit not as robustly as the Cda2-Pep1 vaccine. Finally, seven other peptides were synthesized based on regions in Cda2 predicted to contain promising CD4+ T cell epitopes in BALB/c or C57BL/6 mice. While five peptide vaccines significantly protected BALB/c mice, only one protected C57BL/6 mice. Thus, GP-based vaccines containing a single peptide can protect mice against cryptococcosis. However, given the diversity of human MHC II alleles, a peptide-based Cryptococcus vaccine for use in humans would be challenging and likely need to contain multiple peptide sequences. IMPORTANCE Cryptococcosis, due to infection by fungi of the Cryptococcus neoformans species complex, is responsible for substantial morbidity and mortality in immunocompromised persons, particularly those with AIDS. Cryptococcal vaccines are a public health priority yet are not available for human use. We previously demonstrated mice could be protected from experimental cryptococcosis with vaccines composed of recombinant cryptococcal proteins encased in hollow highly purified yeast cell walls (glucan particles). In this study, we examined one such protective protein, Cda2, and using bioinformatics, we identified a region predicted to stimulate strong T cell responses. A peptide containing this region formulated in glucan particle-based vaccines protected mice as well as the recombinant protein. Other peptide vaccines also protected, including peptides containing sequences from proteins homologous to Cda2. These preclinical mouse studies provide a proof of principle that peptides can be effective as vaccines to protect against cryptococcosis and that bioinformatic approaches can guide peptide selection.

CD82 controls CpG-dependent TLR9 signaling.

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Protection induced by a Francisella tularensis subunit vaccine delivered by glucan particles.

Francisella tularensis is an intracellular pathogen causing the disease tularemia, and an organism of concern to biodefence. There is no licensed vaccine available. Subunit approaches have failed to induce protection, which requires both humoral and cellular immune memory responses, and have been hampered by a lack of understanding as to which antigens are immunoprotective. We undertook a preliminary in silico analysis to identify candidate protein antigens. These antigens were then recombinantly expressed and encapsulated into glucan particles (GPs), purified Saccharomyces cerevisiae cell walls composed primarily of ß-1,3-glucans. Immunological profiling in the mouse was used to down-selection to seven lead antigens: FTT1043 (Mip), IglC, FTT0814, FTT0438, FTT0071 (GltA), FTT0289, FTT0890 (PilA) prior to transitioning their evaluation to a Fischer 344 rat model for efficacy evaluation. F344 rats were vaccinated with the GP protein antigens co-delivered with GP-loaded with Francisella LPS. Measurement of cell mediated immune responses and computational epitope analysis allowed down-selection to three promising candidates: FTT0438, FTT1043 and FTT0814. Of these, a GP vaccine delivering Francisella LPS and the FTT0814 protein was able to induce protection in rats against an aerosol challenge of F. tularensis SchuS4, and reduced organ colonisation and clinical signs below that which immunisation with a GP-LPS alone vaccine provided. This is the first report of a protein supplementing protection induced by LPS in a Francisella vaccine. This paves the way for developing an effective, safe subunit vaccine for the prevention of inhalational tularemia, and validates the GP platform for vaccine delivery where complex immune responses are required for prevention of infections by intracellular pathogens.

Host Control of Fungal Infections: Lessons from Basic Studies and Human Cohorts.

In the last few decades, the AIDS pandemic and the significant advances in the medical management of individuals with neoplastic and inflammatory conditions have resulted in a dramatic increase in the population of immunosuppressed patients with opportunistic, life-threatening fungal infections. The parallel development of clinically relevant mouse models of fungal disease and the discovery and characterization of several inborn errors of immune-related genes that underlie inherited human susceptibility to opportunistic mycoses have significantly expanded our understanding of the innate and adaptive immune mechanisms that protect against ubiquitous fungal exposures. This review synthesizes immunological knowledge derived from basic mouse studies and from human cohorts and provides an overview of mammalian antifungal host defenses that show promise for informing therapeutic and vaccination strategies for vulnerable patients.

A Dectin-1-Caspase-8 Pathway Licenses Canonical Caspase-1 Inflammasome Activation and Interleukin-1ß Release in Response to a Pathogenic Fungus.

Background: Paracoccidioides brasiliensis is equipped with an arsenal of virulence factors that are crucial for causing infection. Our group previously defined the NLRP3 inflammasome as a mediator of P brasiliensis-induced cell damage recognition and induction of effective Th1 immune responses. However, deficiency of caspase-1 only partially reduced interleukin (IL)-1ß levels. Methods: In this study, using chemical inhibitors as well as genetically modified mice, we identify an additional pathway for IL-1ß production in response to P brasiliensis infection. Results: Paracoccidioides brasiliensis initiated caspase-8-mediated IL-1ß production, an event that was necessary for transcriptional priming and posttranslational processing of pro-IL-1ß. Caspase-8 synergizes with the canonical NLRP3 inflammasome pathway to control caspase-1 processing and IL-1ß maturation, providing a regulatory role for caspase-8 in host resistance to in vivo P brasiliensis infection. Conclusions: Taken together, these findings revealed an important role for caspase-8 in the innate immune response of host cells to P brasiliensis infection, demonstrating a connected network between noncanonical and canonical inflammasomes to coordinate IL-1ß production during fungal challenge.

Immunotherapeutic approaches to treatment of fungal diseases.

Fungal infections cause morbidity worldwide and are associated with an unacceptably high mortality despite the availability of antifungal drugs. The incidence of mycoses is rising because of the HIV pandemic and because immunomodulatory drugs are increasingly used to treat autoimmune diseases and cancer. New classes of antifungal drugs have only been partly successful in improving the prognosis for patients with fungal infection. Adjunctive host-directed therapy is therefore believed to be the only option to further improve patient outcomes. Recent advances in the understanding of complex interactions between fungi and host have led to the design and exploration of novel therapeutic strategies in cytokine therapy, vaccines, and cellular immunotherapy, each of which might become viable adjuncts to existing antifungal regimens. In this report, we discuss immunotherapeutic approaches-the rationale behind their design, the challenges in their use, and the progress that is so urgently needed to overcome the devastating effect of fungal diseases.

Properdin Levels in Individuals with Chemotherapy-Induced Neutropenia.

BACKGROUND: Neutrophils produce and carry key components of the alternative pathway (AP) of complement, including properdin (P). The effect of chemotherapy-induced absolute neutropenia on circulating P levels and AP function has not been previously established. METHODS: We prospectively measured free P levels in serum from 27 individuals expected to develop neutropenia after administration of chemotherapy for hematological malignancies in preparation for hematopoietic stem cell transplantation and here describe the relationship between serum P levels and the neutrophil count over time. RESULTS: When the absolute neutrophil count (ANC) was >500 cells/mm3 pre-chemotherapy, P levels were significantly higher than P levels associated with an ANC ≤500 cells/mm3 (median values 8392 ng/mL and 6355 ng/mL, respectively; P = .001). Pairwise comparison between pre-chemotherapy P levels and P levels at initial or last documented neutropenia before recovery showed a significant decline (P < .0001). No correlation was observed between P levels during neutropenia and after recovery of neutropenia in 20 subjects for which postneutropenia samples were obtained. A small but significant (P = .02) decrease in AP hemolytic activity was noted between baseline (preneutropenia) and samples obtained at the onset of neutropenia, but only with low (6.25%) and not higher (12.5 or 25%) serum concentrations. CONCLUSIONS: A decline in P levels and AP activity could contribute to the increased risk of infection in neutropenic patients and warrants further study.

Aspergillus vaccines: Hardly worth studying or worthy of hard study?

Vaccines rank among the greatest advances in the history of public health. Yet, despite the need, there are no licensed vaccines to protect humans against fungal diseases, including aspergillosis. In this focused review, some of the major scientific and logistical challenges to developing vaccines to protect at-risk individuals against aspergillosis are discussed. Approaches that have shown promise in animal models include vaccines that protect against multiple fungal genera and those that are specifically directed to Aspergillus Advances in proteomics and glycomics have facilitated identification of candidate antigens for use in subunit vaccines. Novel adjuvants and delivery systems are becoming available that can skew vaccine responses toward those associated with protection. Immunotherapy consisting of adoptive transfer of Aspergillus-specific T cells to allogeneic hematopoietic transplant recipients has advanced to human testing but is technically difficult and of unproven benefit. While progress has been impressive, much work still needs to be done if vaccines against aspergillosis are to become a reality.

Paradoxical Immune Responses in Non-HIV Cryptococcal Meningitis.

The fungus Cryptococcus is a major cause of meningoencephalitis in HIV-infected as well as HIV-uninfected individuals with mortalities in developed countries of 20% and 30%, respectively. In HIV-related disease, defects in T-cell immunity are paramount, whereas there is little understanding of mechanisms of susceptibility in non-HIV related disease, especially that occurring in previously healthy adults. The present description is the first detailed immunological study of non-HIV-infected patients including those with severe central nervous system (s-CNS) disease to 1) identify mechanisms of susceptibility as well as 2) understand mechanisms underlying severe disease. Despite the expectation that, as in HIV, T-cell immunity would be deficient in such patients, cerebrospinal fluid (CSF) immunophenotyping, T-cell activation studies, soluble cytokine mapping and tissue cellular phenotyping demonstrated that patients with s-CNS disease had effective microbiological control, but displayed strong intrathecal expansion and activation of cells of both the innate and adaptive immunity including HLA-DR+ CD4+ and CD8+ cells and NK cells. These expanded CSF T cells were enriched for cryptococcal-antigen specific CD4+ cells and expressed high levels of IFN-γ as well as a lack of elevated CSF levels of typical T-cell specific Th2 cytokines -- IL-4 and IL-13. This inflammatory response was accompanied by elevated levels of CSF NFL, a marker of axonal damage, consistent with ongoing neurological damage. However, while tissue macrophage recruitment to the site of infection was intact, polarization studies of brain biopsy and autopsy specimens demonstrated an M2 macrophage polarization and poor phagocytosis of fungal cells. These studies thus expand the paradigm for cryptococcal disease susceptibility to include a prominent role for macrophage activation defects and suggest a spectrum of disease whereby severe neurological disease is characterized by immune-mediated host cell damage.

Spectrum and mechanisms of inflammasome activation by chitosan.

Chitosan, the deacetylated derivative of chitin, can be found in the cell wall of some fungi and is used in translational applications. We have shown that highly purified preparations of chitosan, but not chitin, activate the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in primed mouse bone marrow-derived macrophages (BMMΦ), inducing a robust IL-1ß response. In this article, we further define specific cell types that are activated and delineate mechanisms of activation. BMMΦ differentiated to promote a classically activated (M1) phenotype released more IL-1ß in response to chitosan than intermediate or alternatively activated macrophages (M2). Chitosan, but not chitin, induced a robust IL-1ß response in mouse dendritic cells, peritoneal macrophages, and human PBMCs. Three mechanisms for NLRP3 inflammasome activation may contribute: K(+) efflux, reactive oxygen species, and lysosomal destabilization. The contributions of these mechanisms were tested using a K(+) efflux inhibitor, high extracellular potassium, a mitochondrial reactive oxygen species inhibitor, lysosomal acidification inhibitors, and a cathepsin B inhibitor. These studies revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, neither chitosan nor chitin stimulated significant release from unprimed BMMΦ of any of 22 cytokines and chemokines assayed. This study has the following conclusions: 1) chitosan, but not chitin, stimulates IL-1ß release from multiple murine and human cell types; 2) multiple nonredundant mechanisms appear to participate in inflammasome activation by chitosan; and 3) chitin and chitosan are relatively weak stimulators of inflammatory mediators from unprimed BMMΦ. These data have implications for understanding the nature of the immune response to microbes and biomaterials that contain chitin and chitosan.

Cryptococcosis-IRIS is associated with lower cryptococcus-specific IFN-γ responses before antiretroviral therapy but not higher T-cell responses during therapy.

BACKGROUND: Cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS) may be driven by aberrant T-cell responses against cryptococci. We investigated this in human immunodeficiency virus (HIV)-infected patients with treated cryptococcal meningitis (CM) commencing combination antiretroviral therapy (cART). METHODS: Mitogen- and cryptococcal mannoprotein (CMP)-activated (CD25+CD134+) CD4+ T cells and -induced production of interferon-gamma (IFN-γ), IL-10, and CXCL10 were assessed in whole blood cultures in a prospective study of 106 HIV-CM coinfected patients. RESULTS: Patients with paradoxical C-IRIS (n = 27), compared with patients with no neurological deterioration (no ND; n = 63), had lower CMP-induced IFN-γ production in 24-hour cultures pre-cART and 4 weeks post-cART (P = .0437 and .0257, respectively) and lower CMP-activated CD4+ T-cell counts pre-cART (P = .0178). Patients surviving to 24 weeks had higher proportions of mitogen-activated CD4+ T cells and higher CMP-induced CXCL10 and IL-10 production in 24-hour cultures pre-cART than patients not surviving (P = .0053, .0436 and .0319, respectively). C-IRIS was not associated with higher CMP-specific T-cell responses before or during cART. CONCLUSION: Greater preservation of T-cell function and higher CMP-induced IL-10 and CXCL10 production before cART are associated with improved survival while on cART. Lower CMP-induced IFN-γ production pre-cART, but not higher CMP-specific T-cell responses after cART, were risk factors for C-IRIS.

The phenotype of the Cryptococcus-specific CD4+ memory T-cell response is associated with disease severity and outcome in HIV-associated cryptococcal meningitis.

BACKGROUND: Correlates of immune protection in patients with human immunodeficiency virus (HIV)-associated cryptococcal meningitis are poorly defined. A clearer understanding of these immune responses is essential to inform rational development of immunotherapies. METHODS: Cryptococcal-specific peripheral CD4(+) T-cell responses were measured in 44 patients with HIV-associated cryptococcal meningitis at baseline and during follow-up. Responses were assessed following ex vivo cryptococcal mannoprotein stimulation, using 13-color flow-cytometry. The relationships between cryptococcal-specific CD4(+) T-cell responses, clinical parameters at presentation, and outcome were investigated. RESULTS: Cryptococcal-specific CD4(+) T-cell responses were characterized by the production of macrophage inflammatory protein 1α, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α). Conversely, minimal interleukin 4 and interleukin 17 production was detected. Patients surviving to 2 weeks had significantly different functional CD4(+) T-cell responses as compared to those who died. Patients with a response predominantly consisting of IFN-γ or TNF-α production had a 2-week mortality of 0% (0/20), compared with 25% (6/24) in those without this response (P = .025). Such patients also had lower fungal burdens (10 400 vs 390 000 colony-forming units/mL; P < .001), higher cerebrospinal fluid lymphocyte counts (122 vs 8 cells/µL; P < .001), and a trend toward faster rates of clearance of infection. CONCLUSIONS: The phenotype of the peripheral CD4(+) T-cell response to Cryptococcus was associated with disease severity and outcome in HIV-associated cryptococcal meningitis. IFN-γ/TNF-α-predominant responses were associated with survival.

Construction and evaluation of a novel recombinant T cell epitope-based vaccine against Coccidioidomycosis.

Clinical and animal studies of coccidioidomycosis have demonstrated that activated CD4(+) T lymphocytes are essential for protection against this fungal respiratory disease. We previously reported a vaccine against Coccidioides infection which contained three recombinant CD4(+) T cell-reactive proteins and induced a robust, protective immune response in mice. Due to the anticipated high cost of production and clinical assessment of this multivalent vaccine, we generated a single protein which contained immunodominant T cell epitopes of the three polypeptides. Epitopes were initially identified by computational prediction of their ability to bind promiscuously to human major histocompatibility complex class II (MHC II) molecules. Cellular immunoassays confirmed the immunogenicity of the synthesized epitope peptides, while in vitro binding assays revealed a range of peptide affinity for MHC II. A DNA construct was synthesized for bacterial expression of a recombinant protein vaccine which contained five epitopes with the highest affinity for human MHC II, each fused with leader and spacer peptides proposed to optimize epitope processing and presentation to T cell receptors. Recall assays of immune T lymphocytes obtained from human MHC II-expressing HLA-DR4 transgenic mice confirmed that 4 of the 5 epitope peptides were processed. Mice immunized with the epitope-based vaccine admixed with a synthetic oligodeoxynucleotide adjuvant or loaded into yeast glucan particles and then challenged intranasally with Coccidioides showed early lung infiltration of activated T helper-1 (Th1), Th2, and Th17 cells, elevated gamma interferon (IFN-γ) and interleukin (IL)-17 production, significant reduction of fungal burden, and prolongation of survival compared to nonvaccinated mice. This is the first report of an epitope-based vaccine against coccidioidomycosis.

Relative contributions of dectin-1 and complement to immune responses to particulate ß-glucans.

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate ß-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble ß-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.

Dynamic virulence: real-time assessment of intracellular pathogenesis links Cryptococcus neoformans phenotype with clinical outcome.

While a myriad of studies have examined host factors that predispose persons to infection with the opportunistic fungal pathogen Cryptococcus neoformans, comparatively little has been done to examine how virulence factor differences among cryptococcal isolates may impact outcome. In the recent report by Alanio et al. (A. Alanio, M. Desnos-Ollivier, and F. Dromer, mBio 2:e00158-11, 2011), novel flow cytometry-based techniques were employed to demonstrate an association between the phenotype of C. neoformans-macrophage interactions, as measured by phagocytosis and intracellular replication, and patient outcomes, as determined by positive cultures on therapy and survival. These experiments establish that the prognosis of patients with cryptococcosis is influenced by the phenotypic properties of the infecting fungal isolate.

Chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis.

Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance because of frequent natural exposure and their use in medical applications. Depending on the preparation studied and the end point measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. We prepared highly purified chitosan and chitin and examined the capacity of these glycans to stimulate murine macrophages to release the inflammasome-associated cytokine IL-1ß. We found that although chitosan was a potent NLRP3 inflammasome activator, acetylation of the chitosan to chitin resulted in a near total loss of activity. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL-1ß stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. In contrast, chitin is relatively inert.

Robust stimulation of humoral and cellular immune responses following vaccination with antigen-loaded beta-glucan particles.

beta-Glucan particles (GPs) are purified Saccharomyces cerevisiae cell walls treated so that they are primarily beta1,3-d-glucans and free of mannans and proteins. GPs are phagocytosed by dendritic cells (DCs) via the Dectin-1 receptor, and this interaction stimulates proinflammatory cytokine secretion by DCs. As the hollow, porous GP structure allows for high antigen loading, we hypothesized that antigen-loaded GPs could be exploited as a receptor-targeted vaccine delivery system. Ovalbumin (OVA) was electrostatically complexed inside the hollow GP shells (GP-OVA). Incubation of C57BL/6J mouse bone marrow-derived DCs with GP-OVA resulted in phagocytosis, upregulation of maturation markers, and rapid proteolysis of OVA. Compared with free OVA, GP-OVA was >100-fold more potent at stimulating the proliferation of OVA-reactive transgenic CD8(+) OT-I and CD4(+) OT-II T cells, as measured by in vitro [(3)H]thymidine incorporation using DCs as antigen-presenting cells. Next, immune responses in C57BL/6J mice following subcutaneous immunizations with GP-OVA were compared with those in C57BL/6J mice following subcutaneous immunizations with OVA absorbed onto the adjuvant alum (Alum/OVA). Vaccination with GP-OVA stimulated substantially higher antigen-specific CD4(+) T-cell lymphoproliferative and enzyme-linked immunospot (ELISPOT) responses than that with Alum/OVA. Moreover, the T-cell responses induced by GP-OVA were Th1 biased (determined by gamma interferon [IFN-gamma] ELISPOT assay) and Th17 biased (determined by interleukin-17a [IL-17a] ELISPOT assay). Finally, both the GP-OVA and Alum/OVA formulations induced strong secretions of IgG1 subclass anti-OVA antibodies, although only GP-OVA induced secretion of Th1-associated IgG2c antibodies. Thus, the GP-based vaccine platform combines adjuvanticity and antigen delivery to induce strong humoral and Th1- and Th17-biased CD4(+) T-cell responses.

Distinct patterns of dendritic cell cytokine release stimulated by fungal beta-glucans and toll-like receptor agonists.

beta-Glucans derived from fungal cell walls have potential uses as immunomodulating agents and vaccine adjuvants. Yeast glucan particles (YGPs) are highly purified Saccharomyces cerevisiae cell walls composed of beta1,6-branched beta1,3-d-glucan and free of mannans. YGPs stimulated secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in wild-type murine bone marrow-derived myeloid dendritic cells (BMDCs) but did not stimulate interleukin-12p70 (IL-12p70) production. A purified soluble beta1,6-branched beta1,3-d-glucan, scleroglucan, also stimulated TNF-alpha in BMDCs. These two beta-glucans failed to stimulate TNF-alpha in Dectin-1 (beta-glucan receptor) knockout BMDCs. Costimulation of wild-type BMDCs with beta-glucans and specific Toll-like receptor (TLR) ligands resulted in greatly enhanced TNF-alpha production but decreased IL-12p70 production compared with TLR agonists alone. The upregulation of TNF-alpha and downregulation of IL-12p70 required Dectin-1, but not IL-10. Gamma interferon (IFN-gamma) priming did not overcome IL-12p70 reduction by beta-glucans. Similar patterns of cytokine regulation were observed in human monocyte-derived dendritic cells (DCs) costimulated with YGPs and the TLR4 ligand lipopolysaccharide. Finally, costimulation of BMDCs with YGPs and either the TLR9 ligand, CpG, or the TLR2/1 ligand, Pam(3)CSK(4), resulted in upregulated secretion of IL-1alpha and IL-10 and downregulated secretion of IL-1beta, IL-6, and IFN-gamma-inducible protein 10 but had no significant effects on IL-12p40, keratinocyte-derived chemokine, monocyte chemotactic protein 1, or macrophage inflammatory protein alpha, compared with the TLR ligand alone. Thus, beta-glucans have distinct effects on cytokine responses following DC stimulation with different TLR agonists. These patterns of response might contribute to the skewing of immune responses during mycotic infections and have implications for the design of immunomodulators and vaccines containing beta-glucans.