Marie-Pierre Heinen

Assessment of the cutaneous immune response during Arthroderma benhamiae and A. vanbreuseghemii infection using an experimental mouse model

Dermatophytoses are common but poorly understood skin infections. Most in vivo studies have been performed using the guinea pig as the experimental animal model, which has several limitations.

Are Th17 Cells Playing a Role in Immunity to Dermatophytosis

Despite their superficial localization in the skin, pathogenic dermatophytes can induce a complex but still misunderstood immune response in their hosts. The cell-mediated immunity (CMI) is correlated with both clinical recovery and protection against reinfection, and CD4+ T lymphocytes have been recognized as a crucial component of the immune defense against dermatophytes. Before the discovery of the Th17 pathway, CMI was considered to be only dependent of Th1 cells, and thus most studies on the immunology of dermatophytosis have focused on the Th1 pathway. Nevertheless, the fine comparative analysis of available scientific data on immunology of dermatophytosis in one hand and on the Th17 pathway mechanisms involved in opportunistic mucosal fungal infections in the other hand reveals that some key elements of the Th17 pathway can be activated by dermatophytes. Stimulation of the Th17 pathway could occur through the activation of some C-type lectin-like receptors and inflammasome in antigen-presenting cells. The Th17 cells could go back to the affected skin and by the production of signature cytokines could induce the effector mechanisms like the recruitment of polymorphonuclear neutrophils and the synthesis of antimicrobial peptides. In conclusion, besides the Th1 pathway, which is important to the immune response against dermatophytes, there are also growing evidences for the involvement of the Th17 pathway.

Relevant Animal Models in Dermatophyte Research

Dermatophytoses are common superficial fungal infections affecting both humans and animals. They are provoked by filamentous fungi called dermatophytes specialized in the degradation of keratinized structures, which allows them to induce skin, hair and nail infections. Despite their high incidence, little investigation has been performed for the understanding of these infections compared to fungal opportunistic infections and most of the studies were based on in vitro experiments. The development of animal models for dermatophyte research is required to evaluate new treatments against dermatophytoses or to increase knowledge about fungal pathogenicity factors or host immune response mechanisms. The guinea pig has been the most often used animal model to evaluate efficacy of antifungal compounds against dermatophytes, while mouse models were preferred to study the immune response generated during the disease. Here, we review the relevant animal models that were developed for dermatophyte research and we discuss the advantages and disadvantages of the selected species, especially guinea pig and mouse.

Assessment of immunogenicity and protective efficacy of Microsporum canis secreted components coupled to monophosphoryl lipid-A adjuvant in a vaccine study using guinea pigs

Microsporum canis is the most common dermatophyte in pets and is of zoonotic importance but currently there is no effective vaccine available to prevent dermatophytosis. The aim of this work was to assess the immunogenicity and protective efficacy of secreted components (SC) from M. canis adjuvanted with the monophosphoryl lipid-A (MPLA), in a vaccine study using the guinea pig as an experimental model. Animals were vaccinated with either the SC adjuvanted with the MPLA, the MPLA adjuvant alone or PBS three times at two-week intervals, until 42 days prior to M. canis infection. A blind evaluation of dermatophytosis symptoms development and fungal persistence in skin was monitored weekly. The antibody response towards the SC and the levels of Interferon (IFN)γ and Interleukin-4 expressed in peripheral blood mononuclear cells were assessed along or at the end of the study period respectively. The animals that received MPLA had a significantly lower clinical score than those inoculated with PBS. However, no significant difference was observed between the guinea pigs vaccinated with the SC adjuvanted with the MPLA and those having received MPLA alone. The results also showed that vaccination induced a strong antibody response towards the SC and an increase in IFNγ mRNA level. Our results show that the MPLA adjuvant used in this vaccine study can induce per se a partial protection against a M. canis infection. Although they induce a delayed-type hypersensitivity reaction in guinea pigs, the SC do not confer a protection under the present experimental conditions.

Overexpression of TLR-2 and TLR-4 mRNA in feline polymorphonuclear neutrophils exposed to Microsporum canis.

BACKGROUND Polymorphonuclear neutrophils (PMNs), along with macrophages, are the first leukocytes recruited to the site of infection in dermatophytoses and are responsible for the in fine elimination of the fungus. It has been demonstrated that feline PMNs produce pro-inflammatory cytokines after stimulation with Microsporum canis. The activation of these cells results from the recognition of specific PAMPs (pathogen associated molecular patterns) from M. canis by PRRs (pattern recognition receptors) of PMNs. The C-type lectin receptors (CLRs) and toll-like receptors (TLRs) are the two main PRRs in phagocytic cells that recognize fungal components. HYPOTHESIS/OBJECTIVE The aim of this study was to evaluate the expression of TLR-2, TLR-4 and dectin-1 mRNA in feline PMNs exposed to different components from M. canis. METHODS Feline PMNs were stimulated for 2 h or 4 h with either live arthroconidia, heat-killed arthroconidia or secreted components from M. canis. The levels of TLR-2, TLR-4 and dectin-1 mRNA were assessed by RT-qPCR. RESULTS Results showed an increase of TLR-2 and TLR-4 mRNA levels in feline PMNs stimulated with live and heat-killed arthroconidia, but not in those stimulated with the secreted components from M. canis. No significant variation in dectin-1 mRNA expression was observed in PMNs stimulated with the different fungal components. CONCLUSIONS AND CLINICAL IMPORTANCE The overexpression of TLR-2 and TLR-4 mRNAs in stimulated feline PMNs suggests that these receptors are involved in the host immune response through the recognition of M. canis PAMPs.

Development of an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Ringworm Infection in Cattle

ABSTRACT The aim of this study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of ringworm infection in cattle. We used available recombinant forms of Trichophyton rubrum dipeptidyl peptidase V (TruDppV) and T. rubrum leucin aminopeptidase 2 (TruLap2), which are 98% identical to Trichophyton verrucosum orthologues. Field serum samples from 135 cattle with ringworm infection, as confirmed by direct microscopy, fluorescence microscopy, and PCR, and from 55 cattle without any apparent skin lesions or history of ringworm infection that served as negative controls were used. Sensitivities, specificities, and positive and negative predictive values were determined to evaluate the diagnostic value of our ELISA. Overall, the ELISAs based on recombinant TruDppV and TruLap2 discriminated well between infected animals and healthy controls. Highly significant differences (P < 0.0001, Mann-Whitney U test) were noted between optical density values obtained when sera from infected versus control cattle were tested. The ELISA developed for the detection of specific antibodies against DppV gave 89.6% sensitivity, 92.7% specificity, a 96.8% positive predictive value, and a 78.4% negative predictive value. The recombinant TruLap2-based ELISA displayed 88.1% sensitivity, 90.9% specificity, a 95.9% positive predictive value, and a 75.7% negative predictive value. To the best of our knowledge, this is the first ELISA based on recombinant antigens for assessing immune responses to ringworm infection in cattle; it is particularly suitable for epidemiological studies and also for the evaluation of vaccines and/or vaccination procedures.

Th1 and Th17 immune responses act complementarily to optimally control superficial dermatophytosis

Dermatophytoses are among the most common fungal infections worldwide, but little is known about the immune response in them. By comparing Trichophyton benhamiae acute superficial dermatophytosis in wild-type and Rag2-/- mice, we showed that TCR-mediated immunity is critical for fungal clearance and clinical recovery. In WT mice, CD4+ T cells isolated from the skin-draining lymph nodes exhibit both T helper type (Th) 1 and Th17 differentiation during infection, with regard to produced cytokines or mRNA levels of transcription factors. Using IL-17A- and IFN-γ-deficient mice, we showed that IL-17A and IFN-γ are individually dispensable but together contribute to the optimal resolution of dermatophytosis. Furthermore, we generated and infected IL-17A and IFN-γ double-deficient mice and showed that both fungal clearance and clinical recovery were much lower in these mice than in single-deficient mice, suggesting the complementary roles of the two cytokines in dermatophytosis resolution. Thus, our data suggest that TCR-mediated immunity is critical for the optimal control of superficial dermatophytosis and that adaptive immunity is polarized to both Th1 and Th17 responses, with the Th17 antifungal response acting on dermatophyte clearance and the Th1 response being involved in both fungal clearance and Th17-inflammation down-modulation.