Tracing Conidial Fate and Measuring Host Cell Antifungal Activity Using a Reporter of Microbial Viability in the Lung

Abstract

Fluorescence can be harnessed to monitor microbial fate and to investigate functional outcomes of individual microbial cell-host cell encounters at portals of entry in native tissue environments. We illustrate this concept by introducing fluorescent Aspergillus reporter (FLARE) conidia that simultaneously report phagocytic uptake and fungal viability during cellular interactions with the murine respiratory innate immune system. Our studies using FLARE conidia reveal stepwise and cell-type-specific requirements for CARD9 and Syk, transducers of C-type lectin receptor and integrin signals, in neutrophil recruitment, conidial uptake, and conidial killing in the lung. By achieving single-event resolution in defined leukocyte populations, the FLARE method enables host cell profiling on the basis of pathogen uptake and killing and may be extended to other pathogens in diverse model host organisms to query molecular, cellular, and pharmacologic mechanisms that shape host-microbe interactions.

Immunity against fungi

“…Dectin-1/SYK/ CARD9 signaling in NADPH oxidase activity is controversial, as dectin-1-dependent (24) and -independent (42) control of β2 integrin (CD18) activation and the respiratory burst have been reported in vitro. Murine Clec7a -/-and Card9 -/-neutrophils display no cell-intrinsic defect in killing A. fumigatus conidia, unlike p47phox -/-neutrophils (43). These data can be reconciled if the major role of dectin-1/CARD9 is to modulate NADPH oxidase and fungal killing via soluble mediators, rather than by cell-intrinsic activation.…”

“…Loss of individual CLRs results in variable susceptibility to C. albicans (22,23) and A. fumigatus (24,43), in part due to strain-specific differences in CLR activation, as shown for C. albicans (59). Consistent with SYK/CARD9 being central for CLR signal integration, mice with hematopoietic or DC-specific Syk deletion or with global CARD9 deficiency are highly susceptible to C. albicans (29,60) and A. fumigatus (43) challenge.…”

The Carbohydrate Lectin Receptor Dectin-1 Mediates the Immune Response to Exserohilum rostratum

“…Third, there may be Dectin-1-independent pathways including complement (all of the in vivo experiments were performed under conditions with depletion of active complement) that could overcome defects in macrophage responses. Lastly, there are additional immune effector cells, such as dendritic cells and neutrophils, in the in vivo milieu that may generate a response to Exserohilum in a Dectin-1-independent manner utilizing alternative CLRs, such as DC-SIGN or Dectin-2/3 (73,74). A deficiency of caspase recruitment domain-containing protein 9 (Card9) increased susceptibility to the dematiaceous mold Phialophora verrucosa (75,76).…”

Mendelian Genetics of Human Susceptibility to Fungal Infection

“…No candidiasis was seen in these patients (Wang et al 2013b) nor in the majority of the patients with p.Q289X and p.R101C mutations (Lanternier et al 2013), implying that different CARD9 mutations may confer different fungal infection susceptibilities at different anatomical sites. Remarkably, although Card9 is indispensable for control of IA, tuberculosis, and listeriosis in mice Dorhoi et al 2010;Jhingran et al 2012), no infections by molds, intracellular bacteria or mycobacteria have thus far been reported in CARD9-deficient humans. Of note, MALT1 mutations were reported recently to result in an autosomal recessive SCID phenotype with severe bacterial infections and associated mucosal candidiasis of the gastrointestinal tract but no systemic fungal disease (Jabara et al 2013).…”