Description: Blastomyces dermatitidis, a dimorphic fungus and the causative agent of blastomycosis, is widely considered an extracellular pathogen, with little evidence for a facultative intracellular lifestyle. We infected mice with spores, that is, the infectious particle, via the pulmonary route and studied intracellular residence, transition to pathogenic yeast, and replication inside lung cells. Nearly 80% of spores were inside cells at 24 h postinfection with 10 4 spores. Most spores were located inside of alveolar macrophages, with smaller numbers in neutrophils and dendritic cells. Real-time imaging showed rapid uptake of spores into alveolar macrophages, conversion to yeast, and intracellular multiplication during in vitro coculture. The finding of multiple yeast in a macrophage was chiefly due to intracellular replication rather than multiple phagocytic events or fusion of macrophages. Depletion of alveolar macrophages curtailed infection in mice infected with spores and led to a 26-fold reduction in lung CFU by 6 d postinfection versus nondepleted mice. Phase transition of the spores to yeast was delayed in these depleted mice over a time frame that correlated with reduced lung CFU. Spores cultured in vitro converted to yeast faster in the presence of macrophages than in medium alone. Thus, although advanced B. dermatitidis infection may exhibit extracellular residence in tissue, early lung infection with infectious spores reveals its unappreciated facultative intracellular lifestyle.

Description: Vaccine immunity to the endemic mycoses of North America requires Th17 cells, but the pattern recognition receptors and signaling pathways that drive these protective responses have not been defined. We show that C-type lectin receptors exert divergent contributions to the development of antifungal Th17 cells and vaccine resistance against Blastomyces dermatitidis , Histoplasma capsulatum , and Coccidioides posadasii . Acquired immunity to B. dermatitidis requires Dectin-2, whereas vaccination against H. capsulatum and C. posadasii infection depends on innate sensing by Dectin-1 and Dectin-2, but not Mincle. Tracking Ag-specific T cells in vivo established that the Card9 signaling pathway acts indispensably and exclusively on differentiation of Th17 cells, while leaving intact their activation, proliferation, survival, and migration. Whereas Card9 signaling is essential, C-type lectin receptors offer distinct and divergent contributions to vaccine immunity against these endemic fungal pathogens. Our work provides new insight into innate immune mechanisms that drive vaccine immunity and Th17 cells.

Description: By binding to its ligand ICAM-1, LFA-1 is known to mediate both adhesion and costimulatory signaling for T cell activation. The constitutively high LFA-1 cell surface expression of invariant NKT (iNKT) cells has been shown to be responsible for their distinctive tissue homing and residency within ICAM-rich endothelial vessels. However, the functional impact of LFA-1 on the activation of iNKT cells and other innate T lymphocyte subsets has remained largely unexplored. In particular, it is not clear whether LFA-1 contributes to innate-like pathways of T cell activation, such as IFN- secretion in response to IL-12. Using a recombinant ICAM-1-Fc fusion protein to stimulate human iNKT cells in the absence of APCs, we show that LFA-1 engagement enhances their IL-12–driven IFN- production. Surprisingly, exposure to high densities of ICAM-1 was also sufficient to activate iNKT cell cytokine secretion independently of IL-12 and associated JAK/STAT signaling. LFA-1 engagement induced elevated cytoplasmic Ca 2+ and rapid ERK phosphorylation in iNKT cells, and the resulting IFN- secretion was dependent on both of these pathways. Analysis of freshly isolated human PBMC samples revealed that a fraction of lymphocytes that showed elevated LFA-1 cell surface expression produced IFN- in response to plate-bound ICAM-1–Fc. A majority of the responding cells were T cells, with the remainder NK cells. The responding T cells included iNKT cells, MAIT cells, and V2 + T cells. These results delineate a novel integrin-mediated pathway of IFN- secretion that is a shared feature of innate lymphocytes.

Description: Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality within immunocompromised patients. In this study, we examined the potential role of macrophage-inducible C-type lectin (Mincle) for host defense against Pneumocystis . Binding assays implementing soluble Mincle carbohydrate recognition domain fusion proteins demonstrated binding to intact Pneumocystis carinii as well as to organism homogenates, and they purified major surface glycoprotein/glycoprotein A derived from the organism. Additional experiments showed that rats with PCP expressed increased Mincle mRNA levels. Mouse macrophages overexpressing Mincle displayed increased binding to P. carinii life forms and enhanced protein tyrosine phosphorylation. The binding of P. carinii to Mincle resulted in activation of FcR-mediated cell signaling. RNA silencing of Mincle in mouse macrophages resulted in decreased activation of Syk kinase after P. carinii challenge, critical in downstream inflammatory signaling. Mincle-deficient CD4-depleted (Mincle –/– ) mice showed a significant defect in organism clearance from the lungs with higher organism burdens and altered lung cytokine responses during Pneumocystis murina pneumonia. Interestingly, Mincle –/– mice did not demonstrate worsened survival during PCP compared with wild-type mice, despite the markedly increased organism burdens. This may be related to increased expression of anti-inflammatory factors such as IL-1Ra during infection in the Mincle –/– mice. Of note, the P. murina –infected Mincle –/– mice demonstrated increased expression of known C-type lectin receptors Dectin-1, Dectin-2, and MCL compared with infected wild-type mice. Taken together, these data support a significant role for Mincle in Pneumocystis modulating host defense during infection.

Description: Chitin exposure in the lung induces eosinophilia and alternative activation of macrophages and is correlated with allergic airway disease. However, the mechanism underlying chitin-induced polarization of macrophages is poorly understood. In this paper, we show that chitin induces alternative activation of macrophages in vivo but does not do so directly in vitro. We further show that airway epithelial cells bind chitin in vitro and produce CCL2 in response to chitin both in vitro and in vivo. Supernatants of chitin-exposed epithelial cells promoted alternative activation of macrophages in vitro, whereas Ab neutralization of CCL2 in the supernate abolished the alternative activation of macrophages. CCL2 acted redundantly in vivo, but mice lacking the CCL2 receptor, CCR2, showed impaired alternative activation of macrophages in response to chitin, as measured by arginase I, CCL17, and CCL22 expression. Furthermore, CCR2 knockout mice exposed to chitin had diminished reactive oxygen species products in the lung, blunted eosinophil and monocyte recruitment, and impaired eosinophil functions as measured by expression of CCL5, IL-13, and CCL11. Thus, airway epithelial cells secrete CCL2 in response to chitin and CCR2 signaling mediates chitin-induced alternative activation of macrophages and allergic inflammation in vivo.

Description: Fungal infections in CD4 + T cell immunocompromised patients have risen sharply in recent years. Although vaccines offer a rational avenue to prevent infections, there are no licensed fungal vaccines available. Inactivated vaccines are safer but less efficacious and require adjuvants that may undesirably bias toward poor protective immune responses. We hypothesized that reducing the TCR signaling threshold could potentiate antifungal CD8 + T cell responses and immunity to inactivated vaccine in the absence of CD4 + T cells. In this study, we show that CBLB, a negative regulator of TCR signaling, suppresses CD8 + T cells in response to inactivated fungal vaccination in a mouse model of CD4 + T cell lymphopenia. Conversely, Cblb deficiency enhanced both the type 1 (e.g., IFN-) and type 17 (IL-17A) CD8 + T cell responses to inactivated fungal vaccines and augmented vaccine immunity to lethal fungal pneumonia. Furthermore, we show that immunization with live or inactivated vaccine yeast did not cause detectable pathologic condition in Cblb –/– mice. Augmented CD8 + T cell responses in the absence of CBLB also did not lead to terminal differentiation or adversely affect the expression of transcription factors T-bet, Eomes, and RORt. Additionally, our adoptive transfer experiments showed that CBLB impedes the effector CD8 + T cell responses in a cell-intrinsic manner. Finally, we showed that ablation of Cblb overcomes the requirement of HIF-1α for expansion of CD8 + T cells upon vaccination. Thus, adjuvants that target CBLB may augment inactivated vaccines and immunity against systemic fungal infections in vulnerable patients.