Direct Inhibition of T-Cell Responses by the Cryptococcus Capsular Polysaccharide Glucuronoxylomannan

The major virulence factor of the pathogenic fungi Cryptococcus neoformans and C. gattii is the capsule. Glucuronoxylomannan (GXM), the major component of the capsule, is a high-molecular-weight polysaccharide that is shed during cryptococcosis and can persist in patients after successful antifungal therapy. Due to the importance of T cells in the anticryptococcal response, we studied the effect of GXM on the ability of dendritic cells (DCs) to initiate a T-cell response. GXM inhibited the activation of cryptococcal mannoprotein-specific hybridoma T cells and the proliferation of OVA-specific OT-II T cells when murine bone marrow-derived DCs were used as antigen-presenting cells. Inhibition of OT-II T-cell proliferation was observed when either OVA protein or OVA323-339 peptide was used as antigen, indicating GXM did not merely prevent antigen uptake or processing. We found that DCs internalize GXM progressively over time; however, the suppressive effect did not require DCs, as GXM directly inhibited T-cell proliferation induced by anti-CD3 antibody, concanavalin A, or phorbol-12-myristate-13-acetate/ionomycin. Analysis of T-cell viability revealed that the reduced proliferation in the presence of GXM was not the result of increased cell death. GXM isolated from each of the four major cryptococcal serotypes inhibited the proliferation of human peripheral blood mononuclear cells stimulated with tetanus toxoid. Thus, we have defined a new mechanism by which GXM can impart virulence: direct inhibition of T-cell proliferation. In patients with cryptococcosis, this could impair optimal cell-mediated immune responses, thereby contributing to the persistence of cryptococcal infections. SynopsisInfections due to the pathogenic yeast Cryptococcus are a significant cause of morbidity and mortality in persons with impaired T-cell functions, particularly those with AIDS. The major virulence factor of Cryptococcus is its capsule, which is composed primarily of the polysaccharide glucuronoxylomannan (GXM). The capsule not only surrounds the organism but also is shed during cryptococcosis. GXM is taken up by macrophages in vitro and in vivo; however, little is known about the interaction between GXM and dendritic cells, which are the most potent cells capable of activating T cells. Because of the importance of T cells in the anticryptococcal response, the authors investigated the effect of GXM on the ability of dendritic cells to initiate a T-cell response. They found the polysaccharide was internalized by dendritic cells and inhibited antigen-specific T-cell responses. Furthermore, GXM had a direct, inhibitory effect on T-cell proliferation, independent of the effect on dendritic cells. These findings may help explain the persistence of cryptococcal infections and suggest that GXM could be therapeutic in situations where suppression of T-cell responses is desired.National Institutes of Health (R01 AI25780, R01 AI066087, R01 AI37532

Journal Club discussion of Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

This presentation presents the following journal article about COVID-19 for analysis and discussion: Zhou F et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020 Mar 28;395(10229):1054-1062. doi: 10.1016/S0140-6736(20)30566-3

Cooperative Stimulation of Dendritic Cells by Cryptococcus neoformans Mannoproteins and CpG Oligodeoxynucleotides

While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4+ T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.National Institutes of Health (RO1 AI25780, RO1 AI37532, K08 AI 53542

The Case for Adopting the Species Complex Nomenclature for the Etiologic Agents of Cryptococcosis

Cryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii. Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using Cryptococcus neoformans species complex and C. gattii species complex as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion

The Case for Adopting the Species Complex Nomenclature for the Etiologic Agents of Cryptococcosis

Cryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii. Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using Cryptococcus neoformans species complex and C. gattii species complex as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion

Innate sensing of chitin and chitosan

Chitin is the second most common polysaccharide found in nature. It is present in crustacean shells, insect exoskeletons, parasitic nematode eggs and gut linings, and in the cell wall of fungi. The deacetylated derivative of chitin, chitosan, is less common but is particularly evident in certain species of fungi, such as Cryptococcus, and the cyst wall of Entamoeba. How mammals sense and respond to these polymers is not well understood, and conflicting reports on their immunological activity have led to some controversy. Despite this, promising translational applications that exploit the unique properties of chitin and chitosan are being developed

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-gamma 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

A novel vaccine platform using glucan particles for induction of protective responses against Francisella tularensis and other pathogens

Vaccines are considered the bedrock of preventive medicine. However, for many pathogens, it has been challenging to develop vaccines that stimulate protective, long-lasting immunity. We have developed a novel approach using beta-1,3-D-glucans (BGs), natural polysaccharides abundantly present in fungal cell walls, as a biomaterial platform for vaccine delivery. BGs simultaneously provide for receptor-targeted antigen delivery to specialized antigen-presenting cells together with adjuvant properties to stimulate antigen-specific and trained non-specific immune responses. This review focuses on various approaches of using BG particles (GPs) to develop bacterial and fungal vaccine candidates. A special case history for the development of an effective GP tularaemia vaccine candidate is highlighted

All You Need to Know and More about the Diagnosis and Management of Rare Mold Infections

Invasive mold infections caused by molds other than Aspergillus spp. or Mucorales are emerging. The reported prevalences of infection due to these rare fungal pathogens vary among geographic regions, driven by differences in climatic conditions, susceptible hosts, and diagnostic capabilities. These rare molds-Fusarium, Lomentospora, and Scedosporium species and others-are difficult to detect and often show intrinsic antifungal resistance. Now, international societies of medical mycology and microbiology have joined forces and created the Global guideline for the diagnosis and management of rare mould infections: an initiative of the European Confederation of Medical Mycology in cooperation with the International Society for Human and Animal Mycology and the American Society for Microbiology (published in Lancet Infect Dis, https://doi.org/10.1016/S1473-3099(20)30784-2), with the goal of improving the diagnosis, treatment, prevention, and survival of persons with rare mold infections. The guideline provides cutting-edge guidance for the correct utilization and application of established and new diagnostic and therapeutic options

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 \u3e100-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