Antifungal Defense Research Articles

Fungal β-glucans and mannan stimulate peripheral blood mononuclear cells to cytokine production in Syk-dependent manner

There is evidence that major components of the fungi cell wall not only define fungal properties and survival but also are responsible for their biological activities. Some data indicate that structural components of the fungal cell wall exert stimulatory/modulatory effects on immunocompetent cells acting as pathogen-associated molecular patterns (PAMPs). Fungal components can influence the activity of certain immune cell populations by affecting cell maturation and proliferation, promoting phagocytosis, cytotoxic activity, and cell migration, as well as production of various mediators. However, there is little information available concerning the impact of fungal-derived components on peripheral blood mononuclear cell (PBMC) activation. The aim of this study was to determine whether certain fungi-associated molecules, i.e., β-(1,3)-glucans (zymosan and curdlan) and mannan activate in vitro human PBMCs to synthesize cytokines, including chemokines. We documented that PBMCs, in response to stimulation with zymosan, curdlan, and mannan, express cytokines IFN-γ and GM-CSF, and chemokine CCL3, both at protein and transcript levels, as well as cytokine IL-1β and chemokine CXCL8, at mRNA level. Our observations support the idea that fungal-derived components can activate immune cells, including PBMCs, by stimulation of cytokine/chemokine production. A thorough understanding of this interaction is of prime importance since it influence both pathophysiological and immune processes as well as anti-fungal defense mechanisms.

Dectin-1 Promotes Type I and III Interferon Expression to Support Optimal Antifungal Immunity in the Lung.

Pulmonary infections with Aspergillus fumigatus (Af) are a significant cause of invasive fungal disease and lead to high morbidity and mortality in diverse populations throughout the world. Currently available antifungal drugs are often ineffective, thus contributing to unacceptably high mortality rates in patients suffering from invasive fungal infections. The use of cytokines as adjunctive immune therapies holds the promise of significantly improving patient outcomes in the future. In recent studies, we identified an essential role for type I and III interferons as regulators of optimal antifungal responses by pulmonary neutrophils during infection with Af. Although various membrane and cytosolic nucleic acid sensors are known to regulate interferon production in response to viruses, the pathways that regulate the production of these cytokines during fungal infection remain uncovered. In the current study, we demonstrate that dectin-1-mediated recognition of β-glucan on the cell wall of the clinically relevant fungal pathogen Aspergillus fumigatus promotes the activation of a protective cascade of type I and III interferon expression. We further demonstrate that exogenous administration of type I and III interferons can rescue inadequate antifungal responses in dectin-1−/− mice, suggesting the potential therapeutic benefit of these cytokines as activators of antifungal defense in the context of innate defects.

Surfactant Protein D Recognizes Multiple Fungal Ligands: A Key Step to Initiate and Intensify the Anti-fungal Host Defense.

With limited therapeutic options and associated severe adverse effects, fungal infections are a serious threat to human health. Innate immune response mediated by pattern recognition proteins is integral to host defense against fungi. A soluble pattern recognition protein, Surfactant protein D (SP-D), plays an important role in immune surveillance to detect and eliminate human pathogens. SP-D exerts its immunomodulatory activity via direct interaction with several receptors on the epithelial cells lining the mucosal tracts, as well as on innate and adaptive immune cells. Being a C-type lectin, SP-D shows calcium- and sugar-dependent interactions with several glycosylated ligands present on fungal cell walls. The interactome includes cell wall polysaccharides such as 1,3-β-D-glucan, 1,6-β-D-glucan, Galactosaminogalactan Galactomannan, Glucuronoxylomannan, Mannoprotein 1, and glycosylated proteins such as gp45, gp55, major surface glycoprotein complex (gpA). Recently, binding of a recombinant fragment of human SP-D to melanin on the dormant conidia of Aspergillus fumigatus was demonstrated that was not inhibited by sugars, suggesting a likely protein-protein interaction. Interactions of the ligands on the fungal spores with the oligomeric forms of full-length SP-D resulted in formation of spore-aggregates, increased uptake by phagocytes and rapid clearance besides a direct fungicidal effect against C. albicans. Exogenous administration of SP-D showed significant therapeutic potential in murine models of allergic and invasive mycoses. Altered susceptibility of SP-D gene-deficient mice to various fungal infections emphasized relevance of SP-D as an important sentinel of anti-fungal immunity. Levels of SP-D in the serum or lung lavage were significantly altered in the murine models and patients of fungal infections and allergies. Here, we review the cell wall ligands of clinically relevant fungal pathogens and allergens that are recognized by SP-D and their impact on the host defense. Elucidation of the molecular interactions between innate immune humoral such as SP-D and fungal pathogens would facilitate the development of novel therapeutic interventions.

TLR2 and Dectin-1 Signaling in Mouse Hematopoietic Stem and Progenitor Cells Impacts the Ability of the Antigen Presenting Cells They Produce to Activate CD4 T Cells

Microbial recognition by pattern recognition receptors (PRRs) expressed on hematopoietic stem and progenitor cells (HSPCs) not only activates myelopoiesis but also programs the function of the monocytes and macrophages they produce. For instance, changes in HSPC programming modify the ability of macrophages derived from them to produce inflammatory cytokines. While HSPCs exposed to a TLR2 agonist give rise to tolerized macrophages (lower proinflammatory cytokine production), HSPCs treated with Dectin-1 ligands produce trained macrophages (higher proinflammatory cytokine production). However, nothing is known about the impact of HSPC exposure to microbes on the function of antigen presenting cells (APCs). In this study we evaluated whether treatment of murine bone marrow HSPCs with a TLR2 or Dectin-1 ligand impacts the antigen presenting capacity of APCs derived from them in vitro. Following activation with microbial ligands or Candida albicans yeasts, APCs derived from TLR2/Dectin-1-programed HSPCs exhibit altered expression of MHCII (signal 1), co-stimulatory molecules (CD40, CD80 and CD86; signal 2) and cytokines (TNF-α, IL-6, IL-12 p40 and IL-2; signal 3). Moreover, APCs derived from TLR2/Dectin-1-programed HSPCs prime enhanced Th1 and Th17 responses, which are important for antifungal defense, in CD4 T cell cocultures. Overall, these results demonstrate for the first time that microbial detection by bone marrow HSPCs can modulate the adaptive immune response by inducing the production of APCs with an altered phenotype.

Distinct contributions of Type I and Type III IFNs in innate fungal immunity

Abstract Type I (IFN-α/β) and Type III (IFN-λ) interferons are important mediators of antiviral immunity but whether they are required for antifungal defense is unclear. We report that mice defective in Type I and/or Type III IFN receptor expression are highly susceptible to infection with the opportunistic human fungal pathogen Aspergillus fumigatus (Af). Type I IFN (IFNAR) and Type III (IFNLR1) receptor expression on hematopoietic cells was required for antifungal defense while their expression on non-hematopoietic was dispensable. Consistently, mice with gene-specific deletion of IFNLR1 or STAT-1 on granulocytes were highly susceptible to Af. These data demonstrate a requisite and non-redundant role for Type I and Type III IFNs for the activation of antifungal immunity. In addition, we found that Type III IFNs act as important activators of reactive oxygen species generation in antifungal neutrophils, and require early expression of Type I IFN for optimal induction. Importantly, CCR2+ monocytes act as an important source of Type I IFN, and are required for the optimal expression of Type III IFN and fully functional neutrophil antifungal activity. Dysfunctional neutrophil responses in CCR2-depleted mice were rescued by adoptive transfer of pulmonary CCR2+ monocytes or by exogenous administration of IFN-a and IFN-l. Altogether, our data identified Type III IFNs as major regulators of neutrophil activation, and Type I IFNs as early activators of IFN-λ expression and response. Innate antifungal defense is thus coordinated by the actions of Type I and Type III IFNs, a finding that suggests a broader therapeutic potential for these cytokines as activators of antifungal immunity.

De novo indol-3-ylmethyl glucosinolate biosynthesis, and not long-distance transport, contributes to defence of Arabidopsis against powdery mildew.

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.

TAGAP instructs Th17 differentiation by bridging Dectin activation to EPHB2 signaling in innate antifungal response

The TAGAP gene locus has been linked to several infectious diseases or autoimmune diseases, including candidemia and multiple sclerosis. While previous studies have described a role of TAGAP in T cells, much less is known about its function in other cell types. Here we report that TAGAP is required for Dectin-induced anti-fungal signaling and proinflammatory cytokine production in myeloid cells. Following stimulation with Dectin ligands, TAGAP is phosphorylated by EPHB2 at tyrosine 310, which bridges proximal Dectin-induced EPHB2 activity to downstream CARD9-mediated signaling pathways. During Candida albicans infection, mice lacking TAGAP mount defective immune responses, impaired Th17 cell differentiation, and higher fungal burden. Similarly, in experimental autoimmune encephalomyelitis model of multiple sclerosis, TAGAP deficient mice develop significantly attenuated disease. In summary, we report that TAGAP plays an important role in linking Dectin-induced signaling to the promotion of effective T helper cell immune responses, during both anti-fungal host defense and autoimmunity.

Human Neutrophils Produce Antifungal Extracellular Vesicles against Aspergillus fumigatus.

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatusIMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

Overexpression of an apple LysM-containing protein gene, MdCERK1\u20132, confers improved resistance to the pathogenic fungus, Alternaria alternata, in Nicotiana benthamiana

BackgroundLysin motif (LysM)-containing proteins are involved in the recognition of fungal and bacterial pathogens. However, few studies have reported on their roles in the defense responses of woody plants against pathogens. A previous study reported that the apple MdCERK1 gene was induced by chitin and Rhizoctonia solani, and its protein can bind to chitin. However, its effect on defense responses has not been investigated.ResultsIn this study, a new apple CERK gene, designated as MdCERK1–2, was identified. It encodes a protein that shares high sequence identity with the previously reported MdCERK1 and AtCERK1. Its chitin binding ability and subcellular location are similar to MdCERK1 and AtCERK1, suggesting that MdCERK1–2 may play a role in apple immune defense responses as a pattern recognition receptor (PRR). MdCERK1–2 expression in apple was induced by 2 fungal pathogens, Botryosphaeria dothidea and Glomerella cingulate, but not by the bacterial pathogen, Erwinia amylovora, indicating that MdCERK1–2 is involved in apple anti-fungal defense responses. Further functional analysis by heterologous overexpression (OE) in Nicotiana benthamiana (Nb) demonstrated that MdCERK1–2 OE improved Nb resistance to the pathogenic fungus, Alternaria alternata. H2O2 accumulation and callose deposition increased after A. alternata infection in MdCERK1–2 OE plants compared to wild type (WT) and empty vector (EV)-transformed plants. The induced expression of NbPAL4 by A. alternata significantly (p < 0.01, n = 4) increased in MdCERK1–2 OE plants. Other tested genes, including NbNPR1, NbPR1a, NbERF1, and NbLOX1, did not exhibit significant changes after A. alternata infection in OE plants compared to EV or WT plants. OE plants also accumulated more polyphenols after A. alternata infection.ConclusionsHeterologous MdCERK1–2 OE affects multiple defense responses in Nb plants and increased their resistance to fungal pathogens. This result also suggests that MdCERK1–2 is involved in apple defense responses against pathogenic fungi.

Switchable Membrane Remodeling and Antifungal Defense by Metamorphic Chemokine XCL1.

Antimicrobial peptides (AMPs) are a class of molecules which generally kill pathogens via preferential cell membrane disruption. Chemokines are a family of signaling proteins that direct immune cell migration and share a conserved α–β tertiary structure. Recently, it was found that a subset of chemokines can also function as AMPs, including CCL20, CXCL4, and XCL1. It is therefore surprising that machine learning based analysis predicts that CCL20 and CXCL4’s α-helices are membrane disruptive, while XCL1’s helix is not. XCL1, however, is the only chemokine known to be a metamorphic protein which can interconvert reversibly between two distinct native structures (a β-sheet dimer and the α–β chemokine structure). Here, we investigate XCL1’s antimicrobial mechanism of action with a focus on the role of metamorphic folding. We demonstrate that XCL1 is a molecular “Swiss army knife” that can refold into different structures for distinct context-dependent functions: whereas the α–β chemokine structure controls cell migration by binding to G-Protein Coupled Receptors (GPCRs), we find using small angle X-ray scattering (SAXS) that only the β-sheet and unfolded XCL1 structures can induce negative Gaussian curvature (NGC) in membranes, the type of curvature topologically required for membrane permeation. Moreover, the membrane remodeling activity of XCL1’s β-sheet structure is strongly dependent on membrane composition: XCL1 selectively remodels bacterial model membranes but not mammalian model membranes. Interestingly, XCL1 also permeates fungal model membranes and exhibits anti-Candida activity in vitro, in contrast to the usual mode of antifungal defense which requires Th17 mediated cell-based responses. These observations suggest that metamorphic XCL1 is capable of a versatile multimodal form of antimicrobial defense.

Candidalysin Is a Potent Trigger of Alarmin and Antimicrobial Peptide Release in Epithelial Cells.

Host released alarmins and antimicrobial peptides (AMPs) are highly effective as antifungal agents and inducers. Whilst some are expressed constitutively at mucosal tissues, the primary site of many infections, others are elicited in response to pathogens. In the context of Candida albicans, the fungal factors inducing the release of these innate immune molecules are poorly defined. Herein, we identify candidalysin as a potent trigger of several key alarmins and AMPs known to possess potent anti-Candida functions. We also find extracellular ATP to be an important activator of candidalysin-induced epithelial signalling responses, namely epidermal growth factor receptor (EGFR) and MAPK signalling, which mediate downstream innate immunity during oral epithelial infection. The data provide novel mechanistic insight into the induction of multiple key alarmins and AMPs, important for antifungal defences against C. albicans.

Platelets Modulate IFN-γ Production against Candida albicans in Peripheral Blood Mononuclear Cells via Prostaglandins.

Platelets are known to have immunomodulatory properties. They modulate immune responses of leukocytes against various pathogens, including fungi. Candida albicans can cause systemic infection in immunocompromised individuals that is associated with a high mortality and morbidity. In the current study, we explored the role of platelets in antifungal host defense against C. albicans PBMCs were stimulated with heat-killed (HK) C. albicans in the presence or absence of isolated washed platelets. Cytokines were quantified from culture supernatants by ELISA. Inhibition of platelet receptors and cytokine pathways were used to elucidate the mechanisms involved in platelet-leukocyte interaction. In the presence of platelets, PBMCs produced less IFN-γ upon stimulation with HK C. albicans This effect was dependent on the direct contact between platelets and leukocytes but was independent of the platelet GPIb and P-selectin receptors. The attenuation of IFN-γ was not a direct effect on T cells but was dependent on the presence of APC and T cells. Platelets did not modulate the Th-1-polarizing cytokines IL-12 and IL-18. The addition of PG (PGE2) further diminished IFN-γ levels in PBMCs, and supplementation of cells with nonsteroidal anti-inflammatory drugs was able to restore the level of IFN-γ. Overall, we show that modulation of the Th1 response against C. albicans by platelets is dependent on PGs.

IL-23 supports host defense against systemic Candida albicans infection by ensuring myeloid cell survival.

The opportunistic fungal pathogen Candida albicans can cause invasive infections in susceptible hosts and the innate immune system, in particular myeloid cell-mediated immunity, is critical for rapid immune protection and host survival during systemic candidiasis. Using a mouse model of the human disease, we identified a novel role of IL-23 in antifungal defense. IL-23-deficient mice are highly susceptible to systemic infection with C. albicans. We found that this results from a drastic reduction in all subsets of myeloid cells in the infected kidney, which in turn leads to rapid fungal overgrowth and renal tissue injury. The loss in myeloid cells is not due to a defect in emergency myelopoiesis or the recruitment of newly generated cells to the site of infection but, rather, is a consequence of impaired survival of myeloid cells at the site of infection. In fact, the absence of a functional IL-23 pathway causes massive myeloid cell apoptosis upon C. albicans infection. Importantly, IL-23 protects myeloid cells from apoptosis independently of the IL-23-IL-17 immune axis and independently of lymphocytes and innate lymphoid cells. Instead, our results suggest that IL-23 acts in a partially autocrine but not cell-intrinsic manner within the myeloid compartment to promote host protection from systemic candidiasis. Collectively, our data highlight an unprecedented and non-canonical role of IL-23 in securing survival of myeloid cells, which is key for maintaining sufficient numbers of cells at the site of infection to ensure efficient host protection.

Benzoquinone synthesis-related genes of Tribolium castaneum confer the robust antifungal host defense to the adult beetles through the inhibition of conidial germination on the body surface

Insects fight against invading microbial pathogens through various immune-related measures that comprise ‘internal’, ‘external’ as well as ‘social’ immunities. The defenses by external immunity associated with the cuticular integument are supposed to be of particular importance in repelling entomopathogenic fungi that infect host insects transcutaneously. Among such integument-related defenses, external secretions of benzoquinone derivatives typical of tenebrionid beetles have been suggested to play important roles in the antimicrobial defenses. In the present study, by utilizing the experimental infection system composed of the red flour beetle Tribolium castaneum and generalist ascomycete entomopathogens Beauveria bassiana and Metarhizium anisopliae, we performed the functional assays of the three T. castaneum genes whose involvement in benzoquinone synthesis in the adults has been reported, namely GT39, GT62 and GT63. Observations by scanning electron microcopy (SEM) revealed that the conidia of the two fungal species did not germinate on the wild-type adult body surface but did on the pupae. The expression analyses demonstrated that the levels of GT39 and GT62 mRNA increased from middle pupae and reached high in early adults while GT63 did not show a clear adult-biased expression pattern. The RNA interference-based knockdown of any of the three genes in pupae resulted in the adults compromised to the infection of the both fungal species. SEM observations revealed that the gene silencing allowed the conidial germination on the body surface of the knockdown beetles, thereby impairing the robust antifungal defense of adult beetles. Thus, we have provided direct experimental evidence for the functional importance in vivo of these benzoquinone synthesis-related genes that support the antifungal defense of tenebrionid beetles.

Primary immunodeficiencies and invasive fungal infection: when to suspect and how to diagnose and manage.

Invasive fungal infections (IFIs) most often occur secondary to acquired immunodeficiency states such as transplantation, AIDS or immune-modulatory treatment for neoplastic and autoimmune disorders. Apart from these acquired conditions, several primary immunodeficiency disorders (PIDs) can present with IFIs in the absence of iatrogenic immunosuppression. This review highlights recent advances in our understanding of PIDs that cause IFIs, which may help clinicians in the diagnosis and management of such infections. A growing number of PIDs that cause varying combinations of invasive infections by commensal Candida, inhaled molds (primarily Aspergillus), Cryptococcus, Pneumocystis, endemic dimorphic fungi, dermatophytes, and/or agents of phaeohyphomycosis has uncovered the organ- and fungus-specific requirements for effective antifungal host defense in humans. Employing certain diagnostic algorithms tailored to the infecting fungus can facilitate the genetic diagnosis of the underlying PID, which has implications for the optimal management of affected patients. Heightened clinical suspicion is required for the diagnosis of underlying genetic defects in patients who develop IFIs in the absence of acquired immunodeficiency. Early initiation of antifungal therapy followed by long-term secondary prophylaxis is typically needed to achieve remission, but hematopoietic stem-cell transplantation may sometimes be necessary to promote immune restoration and infection control.

Dual Mode of the Saponin Aescin in Plant Protection: Antifungal Agent and Plant Defense Elicitor.

Being natural plant antimicrobials, saponins have potential for use as biopesticides. Nevertheless, their activity in plant–pathogen interaction is poorly understood. We performed a comparative study of saponins' antifungal activities on important crop pathogens based on their effective dose (EC50) values. Among those saponins tested, aescin showed itself to be the strongest antifungal agent. The antifungal effect of aescin could be reversed by ergosterol, thus suggesting that aescin interferes with fungal sterols. We tested the effect of aescin on plant–pathogen interaction in two different pathosystems: Brassica napus versus (fungus) Leptosphaeria maculans and Arabidopsis thaliana versus (bacterium) Pseudomonas syringae pv tomato DC3000 (Pst DC3000). We analyzed resistance assays, defense gene transcription, phytohormonal production, and reactive oxygen species production. Aescin activated B. napus defense through induction of the salicylic acid pathway and oxidative burst. This defense response led finally to highly efficient plant protection against L. maculans that was comparable to the effect of fungicides. Aescin also inhibited colonization of A. thaliana by Pst DC3000, the effect being based on active elicitation of salicylic acid (SA)-dependent immune mechanisms and without any direct antibacterial effect detected. Therefore, this study brings the first report on the ability of saponins to trigger plant immune responses. Taken together, aescin in addition to its antifungal properties activates plant immunity in two different plant species and provides SA-dependent resistance against both fungal and bacterial pathogens.

Interleukin-17 cytokines: Effectors and targets in psoriasis-A breakthrough in understanding and treatment.

The IL-17 cytokine family comprising IL-17A to IL-17F and receptor subunits IL-17RA to IL-17RE represents a genetically ancient intercellular network regulating local tissue homeostasis. Its pivotal role in antifungal defense and its central position in the pathogenesis of inflammatory diseases including psoriasis were discovered only relatively late in the early 2000s. Since the connection of dysregulated IL-17 and psoriasis pathogenesis turned out to be particularly evident, a number of monoclonal antibodies targeting IL-17 pathways have been approved and are used as first line treatment of moderate-to-severe plaque psoriasis and psoriatic arthritis, and further agents are currently in clinical development.

Identification of a novel humoral antifungal defense molecule in the hemolymph of tasar silkworm Antheraea mylitta

Humoral defenses are the major components of insect innate immune system that include the production of several soluble effector molecules from fat body and hemocytes, and released in to the hemolymph upon microbial infection. Hemolymph was collected from the fungal immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, extracted with a mixture of solvent (methanol/glacial acetic acid/water) and fractionated through RP-HPLC. Several fractions were collected, lyophilized and their antifungal activity was tested against Candida albicans. Only the fraction showing strong antifungal activity was further purified via gel filtration chromatography and the purity of active compound was confirmed by thin layer chromatography which showed only single spot after staining with ninhydrin. The molecular mass of this purified compound was determined by high resolution mass spectrometry as 531 Da and analysis of 1H and 13C NMR spectral data along with mass fragmentation pattern indicated the probable structure of the isolated compound as symmetric bis-decanoate derivative. Scanning electron microscopic study revealed that the compound degraded fungal cell wall leading to its lysis and may be the major target for its antifungal activity. These results indicate that presence of this compound in the hemolymph of A.mylitta provides defense against fungal infection.

Interferon-λ orchestrates innate and adaptive mucosal immune responses.

Type III interferon (IFN-λ) was initially thought to have functions similar to those of the type I interferons (IFN-α and IFN-β). New findings have indicated, however, that IFN-λ has a non-redundant role in the innate antiviral, antifungal and antiprotozoal defences of mucosal barriers. In this Review, we highlight recent work showing that IFN-λ inhibits virus dissemination within the body and limits the transmission of respiratory and gastrointestinal viruses to naive hosts. We also discuss findings indicating that IFN-λ can act on neutrophils to prevent invasive pulmonary aspergillosis. We summarize results showing that IFN-λ signalling differs in several respects from IFN-α and IFN-β signalling, particularly in neutrophils. Finally, we discuss new findings indicating that IFN-λ is a potent enhancer of adaptive immune responses in the respiratory mucosa.

Dok3-protein phosphatase 1 interaction attenuates Card9 signaling and neutrophil-dependent antifungal immunity.

Invasive fungal infection is a serious health threat with high morbidity and mortality. Current antifungal drugs only demonstrate partial success in improving prognosis. Furthermore, mechanisms regulating host defense against fungal pathogens remain elusive. Here, we report that the downstream of kinase 3 (Dok3) adaptor negatively regulates antifungal immunity in neutrophils. Our data revealed that Dok3 deficiency increased phagocytosis, proinflammatory cytokine production, and netosis in neutrophils, thereby enhancing mutant mouse survival against systemic infection with a lethal dose of the pathogenic fungus Candida albicans. Biochemically, Dok3 recruited protein phosphatase 1 (PP1) to dephosphorylate Card9, an essential player in innate antifungal defense, to dampen downstream NF-κB and JNK activation and immune responses. Thus, Dok3 suppresses Card9 signaling, and disrupting Dok3-Card9 interaction or inhibiting PP1 activity represents therapeutic opportunities to develop drugs to combat candidaemia.