Interleukin-17A (IL-17A)-producing CD4؉ T helper (Th17) cells have been shown to be essential for defense against pulmonary infection with Coccidioides species. However, we have just begun to identify the required pattern recognition receptors and understand the signal pathways that lead to Th17 cell activation after fungal infection. We previously reported that Card9 ؊/؊ mice vaccinated with formalin-killed spherules failed to acquire resistance to Coccidioides infection. Here, we report that both MyD88 ؊/؊ and Card9 ؊/؊ mice immunized with a live, attenuated vaccine also fail to acquire protective immunity to this respiratory disease. Like Card9 ؊/؊ mice, vaccinated MyD88 ؊/؊ mice revealed a significant reduction in numbers of both Th17 and Th1 cells in their lungs after Coccidioides infection. Both Toll-like receptor 2 (TLR2) and IL-1 receptor type 1 (IL-1r1) upstream of MyD88 have been implicated in Th17 cell differentiation. Surprisingly, vaccinated TLR2 ؊/؊ and wild-type (WT) mice showed similar outcomes after pulmonary infection with Coccidioides, while vaccinated IL-1r1 ؊/؊ mice revealed a significant reduction in the number of Th17 cells in their infected lungs compared to WT mice. Thus, activation of both IL-1r1/MyD88-and Card9-mediated Th17 immunity is essential for protection against Coccidioides infection. Our data also reveal that the numbers of Th17 cells were reduced in IL-1r1 ؊/؊ mice to a lesser extent than in MyD88 ؊/؊ mice, raising the possibility that other TLRs are involved in MyD88-dependent Th17 immunity to coccidioidomycosis. An antimicrobial action of Th17 cells is to promote early recruitment of neutrophils to infection sites. Our data revealed that neutrophils are required for vaccine immunity to this respiratory disease.

There is an emerging interest to develop human vaccines against medically-important fungal pathogens and a need for a preclinical animal model to assess vaccine efficacies and protective correlates. HLA-DR4 (DRB1*0401 allele) transgenic mice express a human major histocompatibility complex class II (MHC II) receptor in such a way that CD4+ T-cell response is solely restricted by this human molecule. In this study HLA-DR4 transgenic mice were immunized with a live-attenuated vaccine (ΔT) and challenged by the intranasal route with 50-70 Coccidioides posadasii spores, a potentially lethal dose. The same vaccination regimen offers 100% survival for C57BL/6 mice. Conversely, ΔT-vaccinated HLA-DR4 mice displayed 3 distinct manifestations of Coccidioides infection including 40% fatal acute (FAD), 30% disseminated (DD) and 30% pulmonary disease (PD). The latter 2 groups of mice had reduced loss of body weight and survived to at least 50 days postchallenge (dpc). These results suggest that ΔT vaccinated HLA-DR4 mice activated heterogeneous immunity against pulmonary Coccidioides infection. Vaccinated HLA-DR4 mice displayed early expansion of Th1 and Th17 cells and recruitment of inflammatory innate cells into Coccidioides-infected lungs during the first 9 dpc. While contraction rates of Th cells and the inflammatory response during 14-35 dpc significantly differed among the 3 groups of vaccinated HLA-DR4 mice. The FAD group displayed a sharply reduced Th1 and Th17 response, while overwhelmingly recruiting neutrophils into lungs during 9-14 days. The FAD group approached moribund by 14 dpc. In contrast, vaccinated HLA-DR4 survivors gradually contracted Th cells and inflammatory response with the greatest rate in the PD group. While vaccinated HLA-DR4 mice are susceptible to Coccidioides infection, they are useful for evaluation of vaccine efficacy and identification of immunological correlates against this mycosis.

Coccidioides is a soil-borne fungal pathogen and causative agent of a human respiratory disease (coccidioidomycosis) endemic to semi-desert regions of southwestern United States, Mexico, Central and South America. Aerosolized arthroconidia inhaled by the mammalian host first undergo conversion to large parasitic cells (spherules, 80–100 μm diameter) followed by endosporulation, a process by which the contents of spherules give rise to multiple endospores. The latter are released upon rupture of the maternal spherules and establish new foci of lung infection. A novel feature of spherule maturation prior to endosporulation is the secretion of a lipid-rich, membranous cell surface layer shed in vivo during growth of the parasitic cells and secretion into liquid culture medium during in vitro growth. Chemical analysis of the culture derived spherule outer wall (SOW) fraction showed that it is composed largely of phospholipids and is enriched with saturated fatty acids, including myristic, palmitic, elaidic, oleic, and stearic acid. NMR revealed the presence of monosaccharide- and disaccharide-linked acylglycerols and sphingolipids. The major sphingolipid components are sphingosine and ceramide. Primary neutrophils derived from healthy C57BL/6 and DBA/2 mice incubated with SOW lipids revealed a significant reduction in fungicidal activity against viable Coccidioides arthroconidia compared to incubation of neutrophils with arthroconidia alone. Host cell exposure to SOW lipids had no effect on neutrophil viability. Furthermore, C57BL/6 mice that were challenged subcutaneously with Coccidioides arthroconidia in the presence of the isolated SOW fraction developed disseminated disease, while control mice challenged with arthroconidia alone by the same route showed no dissemination of infection. We hypothesize that SOW lipids contribute to suppression of inflammatory response to Coccidioides infection. Studies are underway to characterize the immunosuppressive mechanism(s) of SOW lipids.

Introducción: la feohifomicosis es una infección micótica que ocurre especialmente en pacientes inmunosupri­midos y que provoca lesiones cutáneas y, ocasionalmente, en otros órganos. La histopatología, el cultivo y la bio­logía molecular son parte del estudio diagnóstico. El tratamiento incluye la combinación de terapia antimicótica sistémica e intervenciones quirúrgicas. Presentación del caso: hombre de 61 años, diabético y trasplantado renal, con historia de 1 año de lesión ulce­rada en rodilla derecha, no dolorosa y secretante. Histopatológicamente se observaron hifas dematiáceas, con diagnóstico presuntivo de feohifomicosis. Ante la mala evolución clínica a pesar del uso de itraconazol, requirió inicio de anfotericina B y manejo quirúrgico, de donde se aisló en cultivo Cladophialophora spp., confirmando así el diagnóstico. Actualmente está recibiendo voriconazol, con mejoría satisfactoria. Conclusiones: se presenta un caso de feohifomicosis tumoral en un paciente inmunosuprimido, con poca me­joría al uso de terapia sistémica, que requirió combinación con manejo quirúrgico para obtener mejores resul­tados.