Critical role for Ipaf in Pseudomonas aeruginosa‐induced caspase‐1 activation

Abstract

Pseudomonas aeruginosa is an opportunistic Gram-negative human pathogen that is responsible for a broad range of infections in individuals with a variety of predisposing conditions. After infection, P. aeruginosa induces a marked inflammatory response in the host. However the mechanisms involved in bacterium recognition and induction of immune responses are poorly understood. Here we report that the Nod-like receptor family member Ipaf is required for optimal bacterial clearance in an in vivo model of P. aeruginosa lung infection. Further analysis showed that bacterial flagellin was essential for caspase-1 and IL-1b and this activity depended on Ipaf and the adaptor ASC but not TLR5. Notably, P. aeruginosa induced macrophage cell death and this event relied on flagellin and Ipaf but not on ASC. Analysis of Pseudomonas mutants revealed that different amino acid residues of flagellin were critical for sensing by Ipaf and TLR5. Finally, activation of caspase-1 and IL-1b secretion by P. aeruginosa required a functional type III secretion system, but not the effector molecules ExoS, ExoT and ExoY. These results provide new insight into the interaction of P. aeruginosa with host macrophages and suggest that distinct regions of flagellin are sensed by Ipaf and TLR5. Introduction IL-1b plays an important role in the induction of immune responses and in the development of inflammatory disease, fever and septic shock [1]. In response to proinflammatory stimuli including pathogenic bacteria, the IL-1b precursor is induced in monocytes and macrophages and processed into the biologically active IL-1b molecule by caspase-1 [2][3][4][5]. The protease caspase-1 is expressed in monocytes/macrophages as an inactive zymogen that is activated by self cleavage in large multi-protein complexes named 'inflammasomes' [6].The mechanism responsible for activation of caspase-1 in response to microbial stimuli has remained poorly understood. Recent studies have revealed members of the Nod-like receptor (NLR) family as critical components of the inflammasomes by linking microbial sensing to caspase-1 activation [7,8]. For example, Ipaf, an NLR family member and the adaptor ASC have been implicated in activation of caspase-1 in response to Salmonella and Legionella through the cytosolic sensing of flagellin [7,9,10]. Notably, flagellin is also recognized by TLR5 [11]. However, it is unclear whether Ipaf and TLR5 sense identical or distinct regions of flagellin. Similarly, Cryopyrin/Nalp3 is critical for caspase-1 activation and secretion of IL-1b and IL-18 in response to microbial RNA, synthetic purine-like compounds and endogenous urate crystals [12][13][14]. In addition, Cryopyrin regulates caspase-1 activation triggered by exogenous ATP or pore-forming toxins in macrophages stimulated with several TLR agonists [15,16]. Pseudomonas aeruginosa is a flagellated opportunistic Gram-negative human pathogen that is responsible for a broad range of infections in individuals with a variety of predisposing conditions including cystic fibrosis, immu...

Pseudomonas aeruginosa activates caspase 1 through Ipaf

“…While this article was in preparation, other groups also showed that P. aeruginosa activates caspase 1 through Ipaf, T3SS, and flagellin. Some T3SS effectors may modulate this process (30)(31)(32). More than 90% of environmental P. aeruginosa isolates express both flagellin and the Psc T3SS (33,34).…”

Listeria monocytogenes engineered to activate the Nlrc4 inflammasome are severely attenuated and are poor inducers of protective immunity

“…Although ASC is required for activation and secretion of IL-1β and IL-18 downstream of the Nlrc4 inflammasome, it is dispensible for activation of host cell death (Fig. 2B) (28). Therefore, to determine if IL-1β and IL-18 were required for attenuation of L. monocytogenes-L.p.FlaA, we assayed virulence in ASC −/− mice.…”

Cell death in the host response to infection

“…47 Similarly, recently published data show that Pseudomonas aeruginosa requires both Ipaf and ASC to induce IL-1b secretion, but that ASC is dispensable for inducing pyroptosis. 51,86 Conversely, ASC has been shown to induce pyroptosis in the human monocytic THP-1 cell line, engineered to express a GFP-tagged ASC. Upon stimulation with inflammatory agonists, ASC was shown to oligomerize into a single cytoplasmic 'pyroptosome' required for caspase-1 activation and induction of pyroptosis.…”

“…41,49,52,56,66 In some cases, the effector molecule responsible for caspase-1 activation has been identified. For the flagellated bacteria Salmonella typhimurium, 82,83 Legionella pneumophila, 54 and Pseudomonas aeruginosa, 86,91 proper expression of flagellin is required for caspase-1 activation. In addition, purified flagellin is also able to induce caspase-1 activity if introduced into the cytosol through cationic lipids such as DOTAP.…”

“…In addition, purified flagellin is also able to induce caspase-1 activity if introduced into the cytosol through cationic lipids such as DOTAP. 82,83,86 Although stimulating IL-1b and IL-18 secretion, cytosolic flagellin alone is not sufficient to induce cell death. Indeed, activation of caspase-1 in the absence of cell death is seen in virtually all cases of inflammasome activation with purified agonists.…”