Gut-Evolved Candida albicans Induces Metabolic Changes in Neutrophils.

Serial passaging of the human fungal pathogen Candida albicans in the gastrointestinal tract of antibiotics-treated mice selects for virulence-attenuated strains. These gut-evolved strains protect the host from infection by a wide range of pathogens via trained immunity. Here, we further investigated the molecular and cellular mechanisms underlying this innate immune memory. Both Dectin-1 (the main receptor for ß-glucan; a well-described immune training molecule in the fungal cell wall) and Nod2 (a receptor described to mediate BCG-induced trained immunity), were redundant for the protection induced by gut-evolved C. albicans against a virulent C. albicans strain, suggesting that gut-evolved C. albicans strains induce trained immunity via other pathways. Cytometry by time of flight (CyTOF) analysis of mouse splenocytes revealed that immunization with gut-evolved C. albicans resulted in an expansion of neutrophils and a reduction in natural killer (NK) cells, but no significant numeric changes in monocytes, macrophages or dendritic cell populations. Systemic depletion of phagocytes or neutrophils, but not of macrophages or NK cells, reduced protection mediated by gut-evolved C. albicans. Splenocytes and bone marrow cells of mice immunized with gut-evolved C. albicans demonstrated metabolic changes. In particular, splenic neutrophils displayed significantly elevated glycolytic and respiratory activity in comparison to those from mock-immunized mice. Although further investigation is required for fully deciphering the trained immunity mechanism induced by gut-evolved C. albicans strains, this data is consistent with the existence of several mechanisms of trained immunity, triggered by different training stimuli and involving different immune molecules and cell types.

TACI Constrains TH17 Pathogenicity and Protects against Gut Inflammation.

TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) plays critical roles in B cells by promoting immunoglobulin class switching and plasma cell survival. However, its expression and function in T cells remain controversial. We show here that TACI expression can be strongly induced in murine CD4+ T cells in vitro by cytokines responsible for TH17 but not TH1 or TH2 differentiation. Frequencies and numbers of TH17 cells were elevated in TACI-/ - compared with wild-type mice as well as among TACI-/ - versus wild-type CD4+ T cells in mixed bone marrow chimeras, arguing for a T cell-intrinsic effect in the contribution of TACI deficiency to TH17 cell accumulation. TACI-/ - mice were more susceptible to severe colitis induced by dextran sodium sulfate or adoptive T cell transfer, suggesting that TACI negatively regulates TH17 function and limits intestinal inflammation in a cell-autonomous manner. Finally, transcriptomic and biochemical analyses revealed that TACI-/ - CD4+ T cells exhibited enhanced activation of TH17-promoting transcription factors NFAT, IRF4, c-MAF, and JUNB. Taken together, these findings reveal an important role of TACI in constraining TH17 pathogenicity and protecting against gut disease.

Experimental evolution of a fungal pathogen into a gut symbiont.

Gut microbes live in symbiosis with their hosts, but how mutualistic animal-microbe interactions emerge is not understood. By adaptively evolving the opportunistic fungal pathogen Candida albicans in the mouse gastrointestinal tract, we selected strains that not only had lost their main virulence program but also protected their new hosts against a variety of systemic infections. This protection was independent of adaptive immunity, arose as early as a single day postpriming, was dependent on increased innate cytokine responses, and was thus reminiscent of "trained immunity." Because both the microbe and its new host gain some advantages from their interaction, this experimental system might allow direct study of the evolutionary forces that govern the emergence of mutualism between a mammal and a fungus.

The Elusive Anti-Candida Vaccine: Lessons From the Past and Opportunities for the Future.

Candidemia is a bloodstream fungal infection caused by Candida species and is most commonly observed in hospitalized patients. Even with proper antifungal drug treatment, mortality rates remain high at 40-50%. Therefore, prophylactic or preemptive antifungal medications are currently recommended in order to prevent infections in high-risk patients. Moreover, the majority of women experience at least one episode of vulvovaginal candidiasis (VVC) throughout their lifetime and many of them suffer from recurrent VVC (RVVC) with frequent relapses for the rest of their lives. While there currently exists no definitive cure, the only available treatment for RVVC is again represented by antifungal drug therapy. However, due to the limited number of existing antifungal drugs, their associated side effects and the increasing occurrence of drug resistance, other approaches are greatly needed. An obvious prevention measure for candidemia or RVVC relapse would be to immunize at-risk patients with a vaccine effective against Candida infections. In spite of the advanced and proven techniques successfully applied to the development of antibacterial or antiviral vaccines, however, no antifungal vaccine is still available on the market. In this review, we first summarize various efforts to date in the development of anti-Candida vaccines, highlighting advantages and disadvantages of each strategy. We next unfold and discuss general hurdles encountered along these efforts, such as the existence of large genomic variation and phenotypic plasticity across Candida strains and species, and the difficulty in mounting protective immune responses in immunocompromised or immunosuppressed patients. Lastly, we review the concept of "trained immunity" and discuss how induction of this rapid and nonspecific immune response may potentially open new and alternative preventive strategies against opportunistic infections by Candida species and potentially other pathogens.

ß-glucan Exposure on the Fungal Cell Wall Tightly Correlates with Competitive Fitness of Candida Species in the Mouse Gastrointestinal Tract.

Candida albicans is responsible for ~400,000 systemic fungal infections annually, with an associated mortality rate of 46-75%. The human gastrointestinal (GI) tract represents the largest natural reservoir of Candida species and is a major source of systemic fungal infections. However, the factors that control GI colonization by Candida species are not completely understood. We hypothesized that the fungal cell wall would play an important role in determining the competitive fitness of Candida species in the mammalian GI tract. To test this hypothesis, we generated a systematic collection of isogenic C. albicans cell wall mutants and measured their fitness in the mouse GI tract via quantitative competition assays. Whereas a large variation in competitive fitness was found among mutants, no correlation was observed between GI fitness and total levels of individual cell wall components. Similar results were obtained in a set of distantly-related Candida species, suggesting that total amounts of individual cell wall components do not determine the ability of fungi to colonize the GI tract. We then subjected this collection of Candida strains and species to an extensive quantitative phenotypic profiling in search for features that might be responsible for their differences in GI fitness, but found no association with the ability to grow in GI-mimicking and stressful environments or with in vitro and in vivo virulence. The most significant association with GI fitness was found to be the strength of signaling through the Dectin-1 receptor. Using a quantitative assay to measure the amount of exposed ß-glucan on the surface of fungal cells, we found this parameter, unlike total ß-glucan levels, to be strongly predictive of competitive fitness in the mouse GI tract. These data suggest that fungal cell wall architecture, more so than its crude composition, critically determines the ability of fungi to colonize the mammalian GI tract. In particular, recognition of exposed ß-glucan by Dectin-1 receptor appears to severely limit Candida GI fitness and hence represents a promising target to reduce fungal colonization in patients at risks of systemic candidiasis.

Post-genome wide association studies and functional analyses identify association of MPP7 gene variants with site-specific bone mineral density.

Our previous genome-wide association study (GWAS) in a Hong Kong Southern Chinese population with extreme bone mineral density (BMD) scores revealed suggestive association with MPP7, which ranked second after JAG1 as a candidate gene for BMD. To follow-up this suggestive signal, we replicated the top single-nucleotide polymorphism rs4317882 of MPP7 in three additional independent Asian-descent samples (n= 2684). The association of rs4317882 reached the genome-wide significance in the meta-analysis of all available subjects (P(meta)= 4.58 × 10(-8), n= 4204). Site heterogeneity was observed, with a larger effect on spine than hip BMD. Further functional studies in a zebrafish model revealed that vertebral bone mass was lower in an mpp7 knock-down model compared with the wide-type (P= 9.64 × 10(-4), n= 21). In addition, MPP7 was found to have constitutive expression in human bone-derived cells during osteogenesis. Immunostaining of murine MC3T3-E1 cells revealed that the Mpp7 protein is localized in the plasma membrane and intracytoplasmic compartment of osteoblasts. In an assessment of the function of identified variants, an electrophoretic mobility shift assay demonstrated the binding of transcriptional factor GATA2 to the risk allele 'A' but not the 'G' allele of rs4317882. An mRNA expression study in human peripheral blood mononuclear cells confirmed that the low BMD-related allele 'A' of rs4317882 was associated with lower MPP7 expression (P= 9.07 × 10(-3), n= 135). Our data suggest a genetic and functional association of MPP7 with BMD variation.

Phagocytosis of apoptotic cells modulates mesenchymal stem cells osteogenic differentiation to enhance IL-17 and RANKL expression on CD4+ T cells.

Osteogenic differentiation of mesenchymal stem cells (MSC) is important to homeostatic bone remodeling. Infiltration of mesenchymal progenitor cells to inflamed joints has been reported in collagen-induced arthritis murine model and in patients with rheumatoid arthritis (RA). Therapeutic application of MSC in RA has been suggested and under investigation. However, the underlying mechanisms on what triggers the migration of MSC from bone marrow (BM) to inflamed joints and how MSC acts in the joints remains elusive. As hemopoietic stem cells and MSC act reciprocally and excessive apoptotic cells (AC) are observed in the BM of patients with RA, we hypothesize that AC may alter MSC osteogenic differentiation resulting in bone erosion in RA. In this study, we demonstrated for the first time that MSC were able to phagocytose AC and this phagocytosis enhanced MSC osteogenic differentiation. AC-treated MSC under osteogenic differentiation expressed CXC-chemokine receptor (CXCR)-4 and CXCR5, which might enable them to migrate toward the inflamed joints. In addition, AC-treated MSC secreted interleukin (IL)-8, monocyte chemoattractant protein-1, and RANTES, which might induce chemotaxis of CD4+ T cells to the inflamed joints. Interestingly, by coculturing AC-treated MSC under osteogenic differentiation with CD4+ T cells, T helper (Th) 17 cells development was significantly enhanced and these Th17 cells promoted osteoclasts formation and bone resorption. Furthermore, the induction of Th17 cells was dependent on increased IL-6 production from major histocompatibility complex class II-expressing AC-treated MSC under osteogenic differentiation. This data provide a novel insight on the role of AC in modulating MSC osteogenic differentiation and function in inflammatory bone diseases.

Association of JAG1 with bone mineral density and osteoporotic fractures: a genome-wide association study and follow-up replication studies.

Bone mineral density (BMD), a diagnostic parameter for osteoporosis and a clinical predictor of fracture, is a polygenic trait with high heritability. To identify genetic variants that influence BMD in different ethnic groups, we performed a genome-wide association study (GWAS) on 800 unrelated Southern Chinese women with extreme BMD and carried out follow-up replication studies in six independent study populations of European descent and Asian populations including 18,098 subjects. In the meta-analysis, rs2273061 of the Jagged1 (JAG1) gene was associated with high BMD (p = 5.27 x 10(-8) for lumbar spine [LS] and p = 4.15 x 10(-5) for femoral neck [FN], n = 18,898). This SNP was further found to be associated with the low risk of osteoporotic fracture (p = 0.009, OR = 0.7, 95% CI 0.57-0.93, n = 1881). Region-wide and haplotype analysis showed that the strongest association evidence was from the linkage disequilibrium block 5, which included rs2273061 of the JAG1 gene (p = 8.52 x 10(-9) for LS and 3.47 x 10(-5) at FN). To assess the function of identified variants, an electrophoretic mobility shift assay demonstrated the binding of c-Myc to the "G" but not "A" allele of rs2273061. A mRNA expression study in both human bone-derived cells and peripheral blood mononuclear cells confirmed association of the high BMD-related allele G of rs2273061 with higher JAG1 expression. Our results identify the JAG1 gene as a candidate for BMD regulation in different ethnic groups, and it is a potential key factor for fracture pathogenesis.

The interferon gamma gene polymorphism +874 A/T is associated with severe acute respiratory syndrome.

BACKGROUND: Cytokines play important roles in antiviral action. We examined whether polymorphisms of IFN-gamma,TNF-alpha and IL-10 affect the susceptibility to and outcome of severe acute respiratory syndrome (SARS). METHODS: A case-control study was carried out in 476 Chinese SARS patients and 449 healthy controls. We tested the polymorphisms of IFN-gamma,TNF-alpha and IL-10 for their associations with SARS. RESULTS: IFN-gamma +874A allele was associated with susceptibility to SARS in a dose-dependent manner (P < 0.001). Individuals with IFN-gamma +874 AA and AT genotype had a 5.19-fold (95% Confidence Interval [CI], 2.78-9.68) and 2.57-fold (95% CI, 1.35-4.88) increased risk of developing SARS respectively. The polymorphisms of IL-10 and TNF-alpha were not associated with SARS susceptibility. CONCLUSION: IFN-gamma +874A allele was shown to be a risk factor in SARS susceptibility.

Mannose-binding lectin in severe acute respiratory syndrome coronavirus infection.

Little is known about the innate immune response to severe acute respiratory syndrome (SARS) coronavirus (CoV) infection. Mannose-binding lectin (MBL), a key molecule in innate immunity, functions as an ante-antibody before the specific antibody response. Here, we describe a case-control study that included 569 patients with SARS and 1188 control subjects and used in vitro assays to investigate the role that MBL plays in SARS-CoV infection. The distribution of MBL gene polymorphisms was significantly different between patients with SARS and control subjects, with a higher frequency of haplotypes associated with low or deficient serum levels of MBL in patients with SARS than in control subjects. Serum levels of MBL were also significantly lower in patients with SARS than in control subjects. There was, however, no association between MBL genotypes, which are associated with low or deficient serum levels of MBL, and mortality related to SARS. MBL could bind SARS-CoV in a dose- and calcium-dependent and mannan-inhibitable fashion in vitro, suggesting that binding is through the carbohydrate recognition domains of MBL. Furthermore, deposition of complement C4 on SARS-CoV was enhanced by MBL. Inhibition of the infectivity of SARS-CoV by MBL in fetal rhesus kidney cells (FRhK-4) was also observed. These results suggest that MBL contributes to the first-line host defense against SARS-CoV and that MBL deficiency is a susceptibility factor for acquisition of SARS.