Identification of lipophilic ligands of Siglec5 and -14 that modulate innate immune responses.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of cell-surface immune receptors that bind to sialic acid at terminal glycan residues. Siglecs also recognize nonsialic acid ligands, many of which remain to be characterized. Here, we found that Siglec5 and Siglec14 recognize lipid compounds produced by Trichophyton, a fungal genus containing several pathogenic species. Biochemical approaches revealed that the Siglec ligands are fungal alkanes and triacylglycerols, an unexpected finding that prompted us to search for endogenous lipid ligands of Siglecs. Siglec5 weakly recognized several endogenous lipids, but the mitochondrial lipid cardiolipin and the anti-inflammatory lipid 5-palmitic acid-hydroxystearic acid exhibited potent ligand activity on Siglec5. Further, the hydrophobic stretch in the Siglec5 N terminus region was found to be required for efficient recognition of these lipids. Notably, this hydrophobic stretch was dispensable for recognition of sialic acid. Siglec5 inhibited cell activation upon ligand binding, and accordingly, the lipophilic ligands suppressed interleukin-8 (IL-8) production in Siglec5-expressing human monocytic cells. Siglec14 and Siglec5 have high sequence identity in the extracellular region, and Siglec14 also recognized the endogenous lipids. However, unlike Siglec5, Siglec14 transduces activating signals upon ligand recognition. Indeed, the endogenous lipids induced IL-8 production in Siglec14-expressing human monocytic cells. These results indicated that Siglec5 and Siglec14 can recognize lipophilic ligands that thereby modulate innate immune responses. To our knowledge, this is the first study reporting the binding of Siglecs to lipid ligands, expanding our understanding of the biological function and importance of Siglecs in the innate immunity.

A Mouse Model of Trichophyton Inflammation Based on Trichophytin-induced Contact Hypersensitivity.

The prevalence of Trichophyton-induced superficial skin mycosis is very high among human patients. Dermatophytes generally infect the epidermis, especially the stratum corneum, forming scales, hyperkeratosis, and vesicles. The important roles played by the immune system in Trichophyton infection are detection of fungal invasion and elimination of fungi.These immune mechanisms are presumed to involve not only innate immunity but also acquired immunity. Therefore, there is a substantial need for studies on treatment methods based on new basic knowledge, and the elucidation of immunological mechanisms of Trichophyton-induced inflammatory reactions is especially important.However, since Trichophyton cannot colonize on the mouse skin, we tried to develop a model for Trichophyton inflammation induced by trichophytin extracted from Trichophyton mentagrophytes using a method based on contact hypersensitivity.Trichophytin is a crude extract that mainly contains fungal cell wall constituents including ß-glucan and zymosan. In this model, TLR2, TLR4, and dectin-1 were highly expressed, and production of IL-17A and IL23 was observed. This indicates that we succeeded in inducing fungal-specific inflammation in the mice.In this review, we introduce a mouse Trichophyton inflammation model developed to investigate the immunological mechanisms of Trichophyton-induced inflammatory reactions. In addition, we report results of evaluation of anti-inflammatory and anti-itching effects of anti-fungal agents using the inflammation model.

Production of anti-Trichophyton rubrum egg yolk immunoglobulin and its therapeutic potential for treating dermatophytosis.

The aim of this study was to estimate the therapeutic potential of specific egg yolk immunoglobulin (IgY) on dermatophytosis caused by Trichophyton rubrum. The IgY was produced by immunizing hens with cell wall proteins of T. rubrum, extracted from eggs by PEG precipitation and then purified by ammonium sulfate precipitation. The cross-reactivity (CR) with other fungi, growth inhibition on T. rubrum in vitro and therapeutic effect on T. rubrum infection in BALB/C mice of the specific IgY were then evaluated. Anti- T. rubrum cell wall proteins IgY (anti-trCWP IgY) presented a certain degree of cross-reactivity with different fungi. In the in vitro and in vivo activity researches, Anti-trCWP IgY showed a significant dose-dependent growth inhibitory effect on T. rubrum in vitro and a significant dose-dependent therapeutic effect on T. rubrum infection in BALB/C mice.

Trichophyton tonsurans-induced kerion celsi with decreased defensin expression and paradoxically increased interleukin-17A production.

We report a case of kerion celsi due to Trichophyton tonsurans. An 18-year-old male student judo practitioner had alopecic patches, black dots and subcutaneous abscesses on the right temporal region. The damaged hair represented endothrix infection with T. tonsurans, as assessed by mycological examinations. He was treated with oral itraconazole without any therapeutic effect, followed by terbinafine with good effect. A skin biopsy showed neutrophil, lymphocyte and histiocyte infiltration into the dermis and subcutaneous tissue with abscesses around a number of dilated hair follicles. Immunostaining showed that the expression level of human ß-defensin 2 (HBD-2) was decreased in the epidermis of the alopecic and adjacent skin. Because interleukin (IL)-17A generally induces HBD-2 production by epidermal keratinocytes, we also immunohistochemically investigated IL-17A expression. Unexpectedly, many IL-17A-bearing cells were found around destructed hair follicles, indicating that IL-17A expression was not attenuated, but rather increased in the skin lesion. Our case suggests that IL-17A-upregulated antimicrobial peptide expression is disordered in kerion celsi, and severe inflammation with IL-17A may cause tissue damage and resultant scar.

Extracellular Vesicles From the Dermatophyte Trichophyton interdigitale Modulate Macrophage and Keratinocyte Functions.

The release of biomolecules critically affects all pathogens and their establishment of diseases. For the export of several biomolecules in diverse species, the use of extracellular vesicles (EVs) is considered to represent an alternative transport mechanism, but no study to date has investigated EVs from dermatophytes. Here, we describe biologically active EVs from the dermatophyte Trichophyton interdigitale, a causative agent of mycoses worldwide. EV preparations from T. interdigitale were examined using nanoparticle-tracking analysis, which revealed vesicular structures 20-380 nm in diameter. These vesicles induced the production of proinflammatory mediators by bone marrow-derived macrophages (BMDMs) and keratinocytes in a dose-dependent manner, and an addition of the EVs to BMDMs also stimulated the transcription of the M1-polarization marker iNOS (inducible nitric oxide synthase) and diminished the expression of the M2 markers arginase-1 and Ym-1. The observed M1 macrophages' polarization triggered by EVs was abolished in cells obtained from knockout Toll-like receptor-2 mice. Also, the EVs-induced productions of pro-inflammatory mediators were blocked too. Furthermore, the EVs from T. interdigitale enhanced the fungicidal activity of BMDMs. These results suggest that EVs from T. interdigitale can modulate the innate immune response of the host and influence the interaction between T. interdigitale and host immune cells. Our findings thus open new areas of investigation into the host-parasite relationship in dermatophytosis.

Exoproteome Analysis of Human Pathogenic Dermatophyte Species and Identification of Immunoreactive Proteins.

PURPOSE: Increasing incidence of onychomycosis and tinea pedis in humans of industrialized countries together with deep tissue infections are a therapeutic challenge in clinical mycology. For a better understanding of the pathology and immunology of infection, the authors analyze the exoproteomes of three reference strains of the most common clinical dermatophyte species (Trichophyton rubrum, Trichophyton interdigitale, Arthroderma benhamiae) and of Trichophyton strains isolated from affected patients. EXPERIMENTAL DESIGN: Extracellular proteins of those in vitro grown strains are separated via 2D High Performance Electrophoresis and identified by mass spectrometry to find proteins with provoked host immune reactivity. RESULTS: More than 80 secreted proteins including virulence factors such as peptidases and other hydrolases are identified. By Western blotting with respective patient sera, up to 31 proteins with significant antigen-antibody reactions are detected in comparison with control sera, for example, peptidases as well as several oxidoreductases. One protein, beta-glucosidase F2SZI9 seems to be a commonly processed antigen in all Trichophyton infections. CONCLUSIONS AND CLINICAL RELEVANCE: These first global exoproteome data of three dermatophyte species can be a stepping stone on the way to further study the molecular mechanisms of Trichophyton pathogenicity-associated traits. Possible candidates for potential new diagnostic methods or vaccination have to be validated in further investigations.

New insights in dermatophyte research.

Dermatophyte research has renewed interest because of changing human floras with changing socioeconomic conditions, and because of severe chronic infections in patients with congenital immune disorders. Main taxonomic traits at the generic level have changed considerably, and now fine-tuning at the species level with state-of-the-art technology has become urgent. Research on virulence factors focuses on secreted proteases now has support in genome data. It is speculated that most protease families are used for degrading hard keratin during nitrogen recycling in the environment, while others, such as Sub6 may have emerged as a result of ancestral gene duplication, and are likely to have specific roles during infection. Virulence may differ between mating partners of the same species and concepts of zoo- and anthropophily may require revision in some recently redefined species. Many of these questions benefit from international cooperation and exchange of materials. The aim of the ISHAM Working Group Dermatophytes aims to stimulate and coordinate international networking on these fungi.

A Unique Clinicopathological Manifestation of Fungal Infection: A Case Series of Deep Dermatophytosis in Immunosuppressed Patients.

BACKGROUND: Dermatophytes are the most common cause of superficial fungal infections in humans. Deep dermatophytosis, however, is rare, described to date only in isolated case reports, usually in the setting of systemic immunosuppression. OBJECTIVE: To present the 15-year experience of a tertiary dermato-mycology clinic with the diagnosis and treatment of deep dermatophytosis. METHODS: Patients were identified by database search. Clinical, mycological, histological, and treatment data were collected from the medical files. RESULTS: Ten patients were identified: nine after solid-organ transplantation and one undergoing chemotherapy, all diagnosed within 3 years after beginning immunosuppression (average 7.5 months). The infective agent in nine cases was Trichophyton rubrum. All patients presented with concurrent superficial fungal infections. Complete resolution was noted in response to systemic antifungal agents. There was no histological evidence of hair-follicle involvement. LIMITATIONS: The limitations of the study were the retrospective design and the small cohort size. CONCLUSION: This case-series study suggests that deep dermatophytosis is a separate entity, distinct from Majocchi's granuloma. It occurs only in immunocompromised patients and is characterized by discrete nodules, an indolent course, the absence of follicular invasion, and proximity to a superficial dermatophyte infection. Systemic antifungal treatment leads to complete resolution. The urgent need for the treatment of superficial fungal infections in immunocompromised patients is emphasized.

TLR2-/- Mice Display Increased Clearance of Dermatophyte Trichophyton mentagrophytes in the Setting of Hyperglycemia.

Dermatophytosis is one of the most common human infections affecting both immunocompetent individuals and immunocompromised patients, in whom the disease is more aggressive and can reach deep tissues. Over the last decades, cases of deep dermatophytosis have increased and the dermatophyte-host interplay remains poorly investigated. Pattern recognition molecules, such as Toll-like receptors (TLR), play a crucial role against infectious diseases. However, there has been very little research reported on dermatophytosis. In the present study, we investigated the role of TLR2 during the development of experimental deep dermatophytosis in normal mice and mice with alloxan-induced diabetes mellitus, an experimental model of diabetes that exhibits a delay in the clearance of the dermatophyte, Trichophyton mentagrophytes (Tm). Our results demonstrated that inoculation of Tm into the footpads of normal mice increases the expression of TLR2 in CD115+Ly6Chigh blood monocytes and, in hypoinsulinemic-hyperglycemic (HH) mice infected with Tm, the increased expression of TLR2 was exacerbated. To understand the role of TLR2 during the development of murine experimental deep dermatophytosis, we employed TLR2 knockout mice. Tm-infected TLR2-/- and TLR2+/+ wild-type mice exhibited similar control of deep dermatophytic infection and macrophage activity; however, TLR2-/- mice showed a noteworthy increase in production of IFN-γ, IL-10, and IL-17, and an increased percentage of splenic CD25+Foxp3+ Treg cells. Interestingly, TLR2-/- HH-Tm mice exhibited a lower fungal load and superior organization of tissue inflammatory responses, with high levels of production of hydrogen peroxide by macrophages, alongside low TNF-α and IL-10; high production of IL-10 by spleen cells; and increased expansion of Tregs. In conclusion, we demonstrate that TLR2 diminishes the development of adaptive immune responses during experimental deep dermatophytosis and, in a diabetic scenario, acts to intensify a non-protective inflammatory response.

An In Vitro Model for the Study of the Macrophage Response Upon Trichophyton rubrum Challenge.

Diversity in the macrophage models currently employed in immunology studies may lead to opposed results and interpretations. In this study, we aimed to analyze the suitability of J774 macrophage-like cells as a model for the interaction between the dermatophyte Trichophyton rubrum and macrophages. J774 cells were competent in fungal phagocytosis, but succumbed to hyphal growth. Nevertheless, they could also secrete IL-1ß in response to the dermatophyte. On the opposite direction, inflammatory, thioglycollate-induced peritoneal macrophages did not succumb to fungal growth and showed no significant IL-1ß production. The proteomic profiling of these cells uncovered vimentin and plastin-2 as proteins whose abundance was altered by the fungal interaction. Our study indicates that this cell line could be an interesting tool in the investigation of T. rubrum infection biology.

Sensitization and cross-reactions of dermatophyte and Candida albicans allergens in patients with chronic urticaria.

BACKGROUND: Chronic fungal infections are known to exacerbate allergic symptoms, including those of asthma and chronic urticaria (CU). We applied four prepared fungal antigens of Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum, and Candida albicans to examine sensitization to each in subjects with CU and onychomycosis and in healthy subjects, and to evaluate the etiologic role of dermatophytic infection in CU and observe any cross-reactions among these four antigens. METHODS: Participants were divided into four groups, including those with CU with onychomycosis (experiment group), those with onychomycosis without allergic diseases (control group 1), those with CU without fungal infections (control group 2), and a healthy group (control group 3). In all subjects, skin prick tests with the four fungal antigens were performed. Subjects in the experiment group and control group 1 were also submitted to mycologic investigations. RESULTS: The experiment group showed significantly higher rates of positivity than the three control groups to T. rubrum, E. floccosum, and T. mentagrophytes antigens. Control group 1 showed rates higher than those in control groups 2 and 3; no significant difference emerged between control groups 2 and 3. Positivity to the C. albicans antigen did not differ among the four groups. In control group 1, rates of positivity to the three dermatophytic antigens did not differ significantly but did for C. albicans. CONCLUSIONS: Fungal infection seems to be an important determinant of trichophyton hypersensitivity. Cross-reactions among T. mentagrophytes, T. rubrum, and E. floccosum antigens were obvious, but none emerged between the antigens of the three dermatophytes and that of C. albicans.

Production of an anti-dermatophyte monoclonal antibody and its application: immunochromatographic detection of dermatophytes.

Tinea refers to superficial infection with one of three fungal genera-Microsporum, Epidermophyton, or Trichophyton-that are collectively known as dermatophytes. These infections are among the most common diseases worldwide and cause chronic morbidity. They are usually diagnosed by direct microscopy and fungal culture, which are burdensome to perform in the clinical setting. To supplement conventional methods, we developed a new method that employs an immunochromatography test for detection of dermatophyte infections. First, anti-Trichophyton monoclonal antibodies (mAb) were produced in mice using a Trichophyton allergen solution as an immunogen. The mAb specificity was assessed by immunostaining alcohol fixed slide cultures and formalin fixed paraffin-embedded microbial samples. Both alcohol- and formalin-fixed samples of all seven species of Trichophyton tested displayed positive immunostaining. Immunochromatography test strips were created using the anti-Trichophyton mAb. The efficiency of the test strip was assessed in patients diagnosed with tinea unguium and in healthy volunteers. Of the 20 patient nails tested, 19 tested positive and one tested negative, whereas of the 17 volunteer nails, only one tested positive. However, KOH microscopic examination of the volunteer nail that tested positive revealed the existence of Trichophyton hyphae. Although the number of nails assayed was small, since the assay had a sensitivity of 95.0% (19/20) and a specificity of 94.1% (16/17), the obtained results were considered to be promising. Thus, while further investigation with a greater number of samples is necessary, this method could potentially be employed as a new diagnostic tool for Trichophyton in the future.

Dectin-1 and Dectin-2 promote control of the fungal pathogen Trichophyton rubrum independently of IL-17 and adaptive immunity in experimental deep dermatophytosis.

Dermatophytoses are chronic fungal infections, the main causative agent of which is Trichophyton rubrum (T. rubrum). Despite their high occurrence worldwide, the immunological mechanisms underlying these diseases remain largely unknown. Here, we uncovered the C-type lectin receptors, Dectin-1 and Dectin-2, as key elements in the immune response to T. rubrum infection in a model of deep dermatophytosis. In vitro, we observed that deficiency in Dectin-1 and Dectin-2 severely compromised cytokine production by dendritic cells. In vivo, mice lacking Dectin-1 and/or Dectin-2 showed an inadequate pro-inflammatory cytokine production in response to T. rubrum infection, impairing its resolution. Strikingly, neither adaptive immunity nor IL-17 response were required for fungal clearance, highlighting innate immunity as the main checkpoint in the pathogenesis of T. rubrum infection.

Possible Role of Trichophytin Antigen in Inducing Impaired Immunological Clearance of Fungus in Onychomycosis.

The immunology of onychomycosis is poorly understood. Th1 and Th17 are the principal effector cells responsible for protective immunity against fungi, while it is assumed that Th2 responses are associated with deleterious effects. The study was conducted to appraise the role of interleukin-6 (IL-6), transforming growth factor ß (TGF-ß) and immunoglobulin E (IgE) in onychomycosis patients and to study skin reactivity to trichophytin antigen in them. Serum samples of 60 cases of chronic onychomycosis and 30 healthy controls were assayed for serum IgE, IL-6 and TGF-ß levels using specific immunoassay kits; 0.01 ml of trichophytin antigen, Candida antigen and phosphate-buffered saline using separate syringes were injected intradermal at three independent sites of the forearm in cases and controls. Serum IL-6 levels were significantly lower in cases as compared to controls, while serum TGF-ß levels in both cases and controls were comparable. Serum IgE levels in cases were significantly higher when compared with controls. Thirty-eight patients showed immediate hypersensitivity response to trichophytin antigen, while none showed delayed hypersensitivity reaction to trichophytin antigen. Constant fungal antigenic stimuli induce a state of anergy as indicated by low serum IL-6 levels and the absence of delayed hypersensitivity reaction to trichophytin antigen in cases, leading to chronicity of infection. High total IgE may indicate a high probability of prior fungal sensitization.

IL-1 signaling inhibits Trichophyton rubrum conidia development and modulates the IL-17 response in vivo.

Dermatophytosis are one of the most common fungal infections in the world. They compromise keratinized tissues and the main etiological agent is Trichophyton rubrum. Macrophages are key cells in innate immunity and prominent sources of IL-1ß, a potent inflammatory cytokine whose main production pathway is by the activation of inflammasomes and caspase-1. However, the role of inflammasomes and IL-1 signaling against T.rubrum has not been reported. In this work, we observed that bone marrow-derived macrophages produce IL-1ß in response to T.rubrum conidia in a NLRP3-, ASC- and caspase-1-dependent fashion. Curiously, lack of IL-1 signaling promoted hyphae development, uncovering a protective role for IL-1ß in macrophages. In addition, mice lacking IL-1R showed reduced IL-17 production, a key cytokine in the antifungal defense, in response to T.rubrum. Our findings point to a prominent role of IL-1 signaling in the immune response to T.rubrum, opening the venue for the study of this pathway in other fungal infections.

Trichophyton rubrum conidia modulate the expression and transport of Toll-like receptor 2 in HaCaT cell.

Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that T. rubrum can modulate the innate immune responses of host cells, which result in the failure of host cells to recognize T. rubrum and initiate effective immune responses. In this study we show how T. rubrum conidia modulate the expression and transport of Toll-like receptor 2 in HaCaT cell. Flow cytometric analysis showed that the surface and total expression of Toll-like receptor 2 were upregulated at the very early stage when keratinocytes were exposed to T. rubrum conidia regardless of the dose, and the upregulation of surface TLR2 was much more significant than that of total TLR2. Moreover, TLR2 expression was suppressed after upregulation in the initial stage of T. rubrum exposure, and the decrease of total TLR2 was earlier than that of surface TLR2. Our results suggest that in the early stage, TLR2 of keratinocytes were upregulated and transported to the cell surface. After then, the expression of TLR2 was suppressed by T. rubrum conidia.

Dermatophytes activate skin keratinocytes via mitogen-activated protein kinase signaling and induce immune responses.

Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation.