Plasmacytoid dendritic cells (pDCs) were initially considered as critical for innate immunity to viruses. However, our group has shown that pDCs bind to and inhibit the growth of Aspergillus fumigatus hyphae and that depletion of pDCs renders mice hypersusceptible to experimental aspergillosis. In this study, we examined pDC receptors contributing to hyphal recognition and downstream events in pDCs stimulated by A. fumigatus hyphae. Our data show that Dectin-2, but not Dectin-1, participates in A. fumigatus hyphal recognition, TNF-α and IFN-α release, and antifungal activity. Moreover, Dectin-2 acts in cooperation with the FcRγ chain to trigger signaling responses. In addition, using confocal and electron microscopy we demonstrated that the interaction between pDCs and A. fumigatus induced the formation of pDC extracellular traps (pETs) containing DNA and citrullinated histone H3. These structures closely resembled those of neutrophil extracellular traps (NETs). The microarray analysis of the pDC transcriptome upon A. fumigatus infection also demonstrated up-regulated expression of genes associated with apoptosis as well as type I interferon-induced genes. Thus, human pDCs directly recognize A. fumigatus hyphae via Dectin-2; this interaction results in cytokine release and antifungal activity. Moreover, hyphal stimulation of pDCs triggers a distinct pattern of pDC gene expression and leads to pET formation. While plasmacytoid dendritic cells (pDCs) are known to be important immune cells involved in protection from viruses and tumors, their role in protection against fungal infections is less clear. Our laboratory has been studying the interplay between pDCs and the fungal pathogen, Aspergillus fumigatus. Our previous work demonstrated that human pDCs bind to and inhibit the growth of A. fumigatus hyphae. Moreover, depletion of pDCs rendered mice very susceptible to experimental infection with A. fumigatus. Here, we show that Dectin-2, a receptor on pDCs, recognizes A. fumigatus hyphae and contributes to cytokine release and antifungal activity. In addition, using confocal and electron microscopy, we demonstrate that upon contact with hyphae, some human pDCs die and form antimicrobial structures called extracellular traps. Finally, using microarrays, we analyzed human pDC gene expression upon A. fumigatus infection and found distinct patterns including the activation of genes previously associated with viral infections and apoptosis. These results provide new insights into the mechanisms by which pDCs might help the immune system when confronted with a fungal invader.

To study the role of TLR2 in a experimental model of chronic pulmonary infection, TLR2-deficient and wild-type mice were intratracheally infected with Paracoccidioides brasiliensis, a primary fungal pathogen. Compared with control, TLR2−/− mice developed a less severe pulmonary infection and decreased NO synthesis. Equivalent results were detected with in vitro-infected macrophages. Unexpectedly, despite the differences in fungal loads both mouse strains showed equivalent survival times and severe pulmonary inflammatory reactions. Studies on lung-infiltrating leukocytes of TLR2−/− mice demonstrated an increased presence of polymorphonuclear neutrophils that control fungal loads but were associated with diminished numbers of activated CD4+ and CD8+ T lymphocytes. TLR2 deficiency leads to minor differences in the levels of pulmonary type 1 and type 2 cytokines, but results in increased production of KC, a CXC chemokine involved in neutrophils chemotaxis, as well as TGF-β, IL-6, IL-23, and IL-17 skewing T cell immunity to a Th17 pattern. In addition, the preferential Th17 immunity of TLR2−/− mice was associated with impaired expansion of regulatory CD4+CD25+FoxP3+ T cells. This is the first study to show that TLR2 activation controls innate and adaptive immunity to P. brasiliensis infection. TLR2 deficiency results in increased Th17 immunity associated with diminished expansion of regulatory T cells and increased lung pathology due to unrestrained inflammatory reactions.

In infectious diseases, the enzyme indolemine 2,3 dioxygenase (IDO) that catalyzes the tryptophan (Trp) degradation along the kynurenines (Kyn) pathway has two main functions, the control of pathogen growth by reducing available Trp and immune regulation mediated by the Kyn-mediated expansion of regulatory T cells via Aryl hydrocarbon receptor (AhR). In pulmonary paracoccidioidomycosis caused by the dimorphic fungus Paracoccidioides brasiliensis, IDO was shown to control the disease severity of both resistant and susceptible mice to the infection, however, only in resistant mice IDO is induced by TGF- signaling that confers a stable tolerogenic phenotype to DCs. Additionally, in pulmonary PCM the tolerogenic function of plasmacytoid dendritic cells (pDCs) was linked to the IDO activity. To further evaluate the function of IDO in pulmonary PCM, IDO deficient (IDO-/-) C57BL/6 mice were intratracheally infected with P. brasiliensis yeasts and the infection analyzed at three post-infection periods regarding several parameters of disease severity and immune response. The fungal loads and tissue pathology of IDO-/- mice were higher than their wild type (WT) controls resulting in increased mortality rates. DCs from infected IDO-/- mice expressed elevated levels of costimulatory molecules and cytokine IL-6 associated with reduced production of IL-12, TNF-, IL-1 TGF- and IL-10. This response was concomitant with a marked reduction in AhR production. The absence of IDO expression caused an increased influx of activated Th17 cells to the lungs with a simultaneous reduction in Th1 and Treg cells. Accordingly, the suppressive cytokines IL-10, TGF-, IL-27 and IL-35 appeared in reduced levels in the lungs of IDO-/- mice. Interestingly, in the lungs of IDO-/- mice an upregulated differentiation of innate lymphoid cells from the ILC3 phenotype was accompanied by a decreased expansion of ILC1 and NK cells. In conclusion, the immunological balance mediated by the axis IDO/AhR is fundamental to determine the balance between Th17/Treg cells and control the severity of pulmonary PCM.

Dectin-1, the innate immune receptor that recognizes β-glucan, plays an important role in immunity against fungal pathogens. Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, has a sugar-rich cell wall mainly composed of mannans and glucans. This fact motivated us to use dectin-1-sufficient and -deficient mice to investigate the role of β-glucan recognition in the immunity against pulmonary paracoccidioidomycosis. Initially, we verified that P. brasiliensis infection reinforced the tendency of dectin-1-deficient macrophages to express an M2 phenotype. This prevalent antiinflammatory activity of dectin-1(-/-) macrophages resulted in impaired fungicidal ability, low nitric oxide production, and elevated synthesis of interleukin 10 (IL-10). Compared with dectin-1-sufficient mice, the fungal infection of dectin-1(-/-) mice was more severe and resulted in enhanced tissue pathology and mortality rates. The absence of dectin-1 has also impaired the production of T-helper type 1 (Th1), Th2, and Th17 cytokines and the activation and migration of T cells to the site of infection. Remarkably, dectin-1 deficiency increased the expansion of regulatory T cells and reduced the differentiation of T cells to the IL-17(+) phenotype, impairing the migration of IL-17(+)CD8(+) T cells and polymorphonuclear cells to infected tissues. In conclusion, dectin-1 exerts an important protective role in pulmonary paracoccidioidomycosis by controlling the innate and adaptive phases of antifungal immunity.

Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4⁺CD25⁺Foxp3⁺ Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-β. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4⁺CD25⁺Foxp3⁺ Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4⁺ and CD8⁺ T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25⁺ cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.