Invasive aspergillosis (IA) is a life-threatening disease that occurs in patients with hematological malignancies 
, solid organ transplants 
, or immunodeficiency syndromes or patients receiving immunosuppressive treatment 
. The genus Aspergillus
includes about 200 species, of which 20 have been reported as human pathogens causing opportunistic infections, allergic states and invasive aspergillosis.
is considered as the second most-common causative agent of fungal infection after Candida albicans
. A. fumigatus
grows at physiological temperature (37°C), has a stable haploid genome, and undergoes asexual reproduction, forming conidiospores that are released into the environment. Due to their small size (2–3 µm in diameter), the conidia can penetrate deeply into the respiratory airway by simple inhalation and adhere to epithelial cells before infection starts 
Normally, this fungus is efficiently eliminated by the immune system in healthy individuals; however it can trigger a severe IA responsible for high rates of morbidity and mortality in immunocompromised people 
. In these patients, Aspergillus
spores begin to germinate in the lungs, forming branching hyphal filaments that break off and enter the bloodstream, leading to vascular invasion throughout the body 
. Almost all organs can be infected after fungal dissemination. Co-infection with other pathogens such as cytomegalovirus (CMV) or Candida
is very common and complicates IA, making it harder to cure.
The innate immune response against A. fumigatus
plays a crucial role in controlling infection 
. Several pattern recognition receptors (PRRs) such as Toll-like receptor (TLR)-2, TLR-4 and dectin-1 
have been observed to play a role in recognition and clearance of the fungus 
. These studies have shown that host resistance to A. fumigatus
involves the induction of pro-inflammatory cytokines including INFγ, interleukin (IL)-12, TNFα, and significantly, IL-1β 
. Nevertheless, the immunostimulatory molecule(s) of A. fumigatus
that are recognized by PRRs and the molecular basis for inflammation initiation are still under investigation.
PRRs sensors of conserved motifs expressed on microbial pathogens called “pathogen-associated molecular patterns” (PAMPs) 
. PAMPs stimulate PRRs such as surface-bound and endosomal TLRs, but also dectin-1 and cytosolic NOD-like receptor (NLR) family members. Stimulation of these PRRs (TLR-2, TLR-4, and dectin-1) during infection with A. fumigatus
subsequently leads to activation of transcription factors such as NF-κB, whose translocation into the nucleus stimulates the upregulation of pro-inflammatory cytokines. Secretion of pro-inflammatory cytokines (TNFα, IL-12 and IL-1β) and chemokines (Mip-2 in mice, IL-8 in humans) helps to recruit neutrophils and lymphocytes to the pulmonary infection site and insure clearance of the fungus. Neutrophils and macrophages are the two main cell types responsible for the innate host response against aspergillosis, therefore the risk of infection is higher in subjects presenting an inadequate number or anomalies of these cell types 
The pro-inflammatory cytokine, IL-1β, is synthesized as an inactive cytoplasmic precursor, pro-IL-1β, which is processed into a biologically active, secreted form by caspase- 1, a cysteine protease 
. The latter is synthesized as an inactive form that is self-activated by cleavage, generating an enzymatically active heterodimer composed of 10 and 20 kDa chains 
. Recent studies have implicated members of the NLR family of proteins in the regulation of caspase-1 activation 
. The NLR family is composed of 23 cytosolic proteins, some of which recognize PAMPs. The family includes nucleotide binding oligomerization domain 1 (NOD1), NOD2 
, the NLRP3/cryopyrin/Nalp3 “inflammasome” component 
, and the NLRC4/Ipaf inflammasome component 
Upon infection, stimulation of TLRs or the cytosolic NOD1 or NOD2 receptors activates transcription, synthesis, and secretion of pro-inflammatory cytokines such as INFγ, IL-12, and TNFα 
. Given the key role played by IL-1β in fever and inflammatory disease 
, its production and secretion is tightly controlled and requires typically two separate signals 
. The first signal comes from PAMPs and promotes transcription, production and intracellular accumulation of the immature cytokine. The second signal, usually derived from a “danger signal” (DS), leads to the activation of an inflammasome, activation of caspase-1, and secretion of the mature cytokine. The requirement for two signals thus insures that IL-1β is secreted by macrophages only if they are stimulated by PAMPs and the PAMPs are produced under circumstances that could be viewed as potentially dangerous to the host organism 
. Examples of DSs include host-cell components released from dying, infected or stressed cells such as ATP, adenosine, uric acid, or chromosomal proteins; but they could also be microbial PAMPs that are located in “threatening” locations, such as flagellin in the cytosol of an infected cell 
Several studies have recently described stimulation of the NLRP3 inflammasome in the innate immune response to C. albicans
. These were the first reports to show the involvement of an inflammasome during a fungal infection. However, stimulation of an inflammasome has not been described yet during A. fulmigatus
infection. Although caspase-1 activation during A. fumigatus
infection has not been investigated, studies showing secretion of IL-1β by the human monocyte/macrophage cell line, THP-1, following stimulation by A. fumigatus 
suggested that caspase-1 must be activated in these cells, either directly by the fungal pathogen or in combination with a host-cell derived DS.
The goal of this study was therefore to determine whether A. fumigatus induces IL-1β secretion in a caspase-1 dependent manner by THP-1 cells, and evaluate whether this fungus activates an inflammasome. Our results show that A. fumigatus spores fail to induce caspase-1 activation, unlike hyphal fragments, which upregulate pro-IL-1β synthesis and stimulate caspase-1 activation. Importantly we revealed the requirement of an NLRP3 inflammasome and its adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), in activating caspase-1, thus revealing NLRP3 and ASC as key regulators of inflammation during A. fumigatus infection.