Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans.

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.

The nucleotide receptor STING translocates to the phagosomes to negatively regulate anti-fungal immunity.

STING (stimulator of interferon genes) exerts protective cellular responses to viral infection via induction of interferon production and autophagy. Here, we report the role of STING in modulating the immune responses toward fungal infection. Upon Candida albicans stimulation, STING transited alongside the endoplasmic reticulum (ER) to the phagosomes. In phagosomes, STING directly bound with Src via the N-terminal 18 amino acids of STING, and this binding prevented Src from recruiting and phosphorylating Syk. Consistently, Syk-associated signaling and production of pro-inflammatory cytokines and chemokines were increased in mouse BMDCs (bone-marrow-derived dendritic cells) lacking STING with fungal treatment. STING deficiency improved anti-fungal immunity in systemic C. albicans infection. Importantly, administration of the N-terminal 18-aa (amino acid) peptide of STING improved host outcomes in disseminated fungal infection. Overall, our study identifies a previously unrecognized function of STING in negatively regulating anti-fungal immune responses and offers a potential therapeutic strategy for controlling C. albicans infection.

Molecular and functional in vivo characterisation of murine Dectin-1 isoforms.

Dectin-1 is a C-type lectin-receptor involved in sensing fungi by innate immune cells. Encoded by the Clec7a gene, Dectin-1 exists in two major splice isoforms, Dectin-1a and 1b, which differ in the presence of a membrane-proximal stalk domain. As reported previously, this domain determines degradative routes for Dectin-1a and 1b leading to the generation of a stable N-terminal fragment exclusively from Dectin-1a. Here, we narrow down the responsible part of the stalk and demonstrate the stabilisation of the Dectin-1a N-terminal fragment in tetraspanin-enriched microdomains. C57BL/6 and BALB/c mice show divergent Dectin-1 isoform expression patterns, which are caused by a single nucleotide polymorphism in exon 3 of the Clec7a gene, leading to a non-sense Dectin-1a mRNA in C57BL/6 mice. Using backcrossing, we generated mice with the C57BL/6 Clec7a allele on a BALB/c background and compared these to the parental strains. Expression of the C57BL/6 allele leads to the exclusive presence of the Dectin-1b protein. Furthermore, it was associated with higher Dectin-1 mRNA expression, but less Dectin-1 at the cell surface according to flow cytometry. In neutrophils, this altered ROS production induced by Dectin-1 model ligands, while cellular responses in macrophages and dendritic cells were not significantly influenced by the Dectin-1 isoform pattern.

Dectin-2-mediated signaling triggered by the cell wall polysaccharides of Cryptococcus neoformans.

Cryptococcus neoformans is rich in polysaccharides of the cell wall and capsule. Dectin-2 recognizes high-mannose polysaccharides and plays a central role in the immune response to fungal pathogens. Previously, we demonstrated Dectin-2 was involved in the activation of dendritic cells upon stimulation with C. neoformans, suggesting the existence of a ligand recognized by Dectin-2. In the present study, we examined the cell wall structures of C. neoformans contributing to the Dectin-2-mediated activation of immune cells. In a NFAT-GFP reporter assay of the reported cells expressing Dectin-2, the lysates, but not the whole yeast cells, of an acapsular strain of C. neoformans (Cap67) delivered Dectin-2-mediated signaling. This activity was detected in the supernatant of ß-glucanase-treated Cap67 and more strongly in the semi-purified polysaccharides of this supernatant using ConA-affinity chromatography (ConA-bound fraction), in which a large amount of saccharides, but not protein, were detected. Treatment of this supernatant with periodic acid and the addition of excessive mannose, but not glucose or galactose, strongly inhibited this activity. The ConA-bound fraction of the ß-glucanase-treated Cap67 supernatant was bound to Dectin-2-Fc fusion protein in a dose-dependent manner and strongly induced the production of interleukin-12p40 and tumour necrosis factor-α by dendritic cells; this was abrogated under the Dectin-2-deficient condition. Finally, 98 kDa mannoprotein (MP98) derived from C. neoformans showed activation of the reporter cells expressing Dectin-2. These results suggested that a ligand with mannose moieties may exist in the cell walls and play a critical role in the activation of dendritic cells during infection with C. neoformans.

Innate immune signal transduction pathways to fungal infection: Components and regulation.

Candida species are significant causes of mucosal and systemic infections in immune compromised populations, including HIV-infected individuals and cancer patients. Drug resistance and toxicity have limited the use of anti-fungal drugs. A good comprehension of the nature of the immune responses to the pathogenic fungi will aid in the developing of new approaches to the treatment of fungal diseases. In recent years, extensive research has been done to understand the host defending systems to fungal infections. In this review, we described how pattern recognition receptors senses the cognate fungal ligands and the cellular and molecular mechanisms of anti-fungal innate immune responses. Furthermore, particular focus is placed on how anti-fungal signal transduction cascades are being activated for host defense and being modulated to better treat the infections in terms of immunotherapy. Understanding the role that these pathways have in mediating host anti-fungal immunity will be crucial for future therapeutic development.

DOCK2 regulates antifungal immunity by regulating RAC GTPase activity.

Fungal infections cause ~1.5 million deaths each year worldwide, and the mortality rate of disseminated candidiasis currently exceeds that of breast cancer and malaria. The major reasons for the high mortality of candidiasis are the limited number of antifungal drugs and the emergence of drug-resistant species. Therefore, a better understanding of antifungal host defense mechanisms is crucial for the development of effective preventive and therapeutic strategies. Here, we report that DOCK2 (dedicator of cytokinesis 2) promotes indispensable antifungal innate immune signaling and proinflammatory gene expression in macrophages. DOCK2-deficient macrophages exhibit decreased RAC GTPase (Rac family small GTPase) activation and ROS (reactive oxygen species) production, which in turn attenuates the killing of intracellular fungi and the activation of downstream signaling pathways. Mechanistically, after fungal stimulation, activated SYK (spleen-associated tyrosine kinase) phosphorylates DOCK2 at tyrosine 985 and 1405, which promotes the recruitment and activation of RAC GTPases and then increases ROS production and downstream signaling activation. Importantly, nanoparticle-mediated delivery of in vitro transcribed (IVT) Rac1 mRNA promotes the activity of Rac1 and helps to eliminate fungal infection in vivo. Taken together, this study not only identifies a critical role of DOCK2 in antifungal immunity via regulation of RAC GTPase activity but also provides proof of concept for the treatment of invasive fungal infections by using IVT mRNA.

Current trends to control fungal pathogens: exploiting our knowledge in the host-pathogen interaction.

Human fungal infections remain a major challenge in medicine. Only a limited number of antifungal drugs are available, which are often related to severe adverse effects. In addition, there is an increased emergence related to resistant strains, which makes imperative to understand the host-pathogen interactions as well as to develop alternative treatments. Host innate and adaptive immunity play a crucial role controlling fungal infections; therefore, vaccines are a viable tool to prevent and treat fungal pathogens. Innate immunity is triggered by the interaction between the cell surface pattern recognition receptors (PRRs) and the pathogen-associated molecular patterns (PAMPs). Such an initial immunological response is yet little understood in fungal infections, in part due to the complexity and plasticity of the fungal cell walls. Described host cell-fungus interactions and antigenic molecules are addressed in this paper. Furthermore, antigens found in the cell wall and capsule, including peptides, glycoproteins, glycolipids, and glycans, have been used to trigger specific immune responses, and an increased production of antibodies has been observed when attached to immunogenic molecules. The recent biotechnological advances have allowed the development of vaccines against viral and bacterial pathogens with positive results; therefore, this technology has been applied to develop anti-fungal vaccines. Passive immunization has also emerged as an appealing alternative to treat disseminated mycosis, especially in immunocompromised patients. Those approaches have a long way to be seen in clinical cases. However, all studies discussed here open the possibility to have access to new therapies to be applied alone or in combination with current antifungal drugs. Herein, the state of the art of fungal vaccine developments is discussed in this review, highlighting new advances against Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Paracoccidioides brasiliensis, and Sporothrix spp.

CTL14, a recognition receptor induced in late stage larvae, modulates anti-fungal immunity in cotton bollworm Helicoverpa armigera.

C-type lectin (CTL) is usually considered as pattern recognition receptors in insect innate immunity. Here we found that CTL14 of Helicoverpa armigera was only activated in the fifth instar larvae not in the second instar by entomopathogen Beauveria bassiana infection. Recombinant CTL14 protein was found to form aggregates with zymosan and B. bassiana in vitro. Immunoprecipitation studies demonstrated that CTL14 interacted with serine proteinases (SP), serine proteinase inhibitor (serpin), prophenoloxidases (PPO) and vitellogenin (Vg) in the larval hemolymph. Furthermore, depletion of CTL14 using dsRNA led to dramatic decrease in the expression level of PPO1. Additionally, CTL14 depleted H. armigera decreased the resistance to fungal challenge. Taken together, our study showed the direct involvement of CTL14 in the anti-fungal immunity of H. armigera, which further explained the stronger immune responses in the fifth instar compared to the second instar larvae.

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages.

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/ß or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1-/- mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1-/- macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.

Differential Regulation of Myeloid-Derived Suppressor Cells by Candida Species.

Myeloid-derived suppressor cells (MDSCs) are innate immune cells characterized by their ability to suppress T-cell responses. Recently, we demonstrated that the human-pathogenic fungi Candida albicans and Aspergillus fumigatus induced a distinct subset of neutrophilic MDSCs. To dissect Candida-mediated MDSC induction in more depth, we studied the relative efficacy of different pathogenic non-albicans Candida species to induce and functionally modulate neutrophilic MDSCs, including C. glabrata, C. parapsilosis, C. dubliniensis, and C. krusei. Our data demonstrate that the extent of MDSC generation is largely dependent on the Candida species with MDSCs induced by C. krusei and C. glabrata showing a higher suppressive activity compared to MDSCs induced by C. albicans. In summary, these studies show that fungal MDSC induction is differentially regulated at the species level and differentially affects effector T-cell responses.