Receptors of the innate immune system detect conserved determinants of microbial and viral origin. Activation of these receptors initiates signaling events that culminate in an effective immune response. Recently, the view that innate immune signaling events rely on and operate within a complex cellular infrastructure has become an important framework for understanding the regulation of innate immunity. Compartmentalization within this infrastructure provides the cell with the ability to assign spatial information to microbial detection and regulate immune responses. Several cell biological processes play a role in the regulation of innate signaling responses; at the same time, innate signaling can engage cellular processes as a form of defense or to promote immunological memory. In this review, we highlight these aspects of cell biology in pattern-recognition receptor signaling by focusing on signals that originate from the cell surface, from endosomal compartments, and from within the cytosol.

Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes, and what they sense, remain poorly defined. Here we demonstrate that 35 amino acids from the Cterminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in Naip5. Naip5-deficient mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Taken together, these data clarify the molecular basis for the cytosolic response to flagellin.Inflammasomes are cytosolic multiprotein complexes that are critical regulators of inflammation, and are required for proteolytic activation of the cysteine protease caspase-1 (refs. 1-3). Caspase-1 (A000492; http://www.signaling-gateway.org/molecule/query?afcsid=A000492) is itself required for the proteolytic processing and release of inflammatory cytokines such as interleukin 1β (IL-1β) and IL-18, as well as for induction of a necrotic-like cell death called pyroptosis1-3. The molecular components and structures of inflammasomes remain poorly defined. It is believed that multiple distinct inflammasomes may exist, each containing a key scaffold protein of the NLR (nucleotide-binding domain, leucine-rich repeat) superfamily that confers specificity for particular microbial products. For example, NLR proteins of the NLRP1 family (also called NALP1) appear to activate the inflammasome in response to anthrax lethal toxin4 and bacterial muramyl dipeptide5. In contrast, the NLR protein NLRP3 (also called NALP3 or cryopyrin) has been proposed to sense a wide range of stimuli including bacterial RNA6, viral DNA7, uric acid crystals8, muramyl dipeptide9,10, nigericin11, amyloid-beta12, and other irritants13-16. There is at present no molecular explanation for how a single NLR protein can NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript be activated by all these microbial products and the precise molecular nature of what is sensed by any inflammasome remains undefined.The inflammasome containing the NLR protein IPAF (also called NLRC4) is one of the best characterized inflammasomes, and has been proposed by several groups to respond to the presence of flagellin in the cytosol17-19. Flagellin-deficient mutants of Salmonella typhimurium and Legionella pneumophila are defective in IPAF-dependent inflammasome activation, and flagellin, purified from or expressed in bacteria, triggers IPAF-dependent caspase-1 activation when delivered to the cytosol of macrophages by use of a pore-forming toxin (listeriolysin O (LLO)) or transfection reagents17-21. It was proposed that during natural infections, flagellin triggers inflammasome activation upon secretion into the host cytosol via bacterial type III/IV secretion systems17-21. However, doubts have been expressed as to w...

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Bacterial and viral mRNAs are often polycistronic. Akin to alternative splicing, alternative translation of polycistronic messages is a mechanism to generate protein diversity and regulate gene function. Although a few examples exist, the use of polycistronic messages in mammalian cells is not widely appreciated. Here we report an example of alternative translation as a means of regulating innate immune signaling. MAVS, a regulator of antiviral innate immunity, is expressed from a bicistronic mRNA encoding a second protein, miniMAVS. This truncated variant interferes with interferon production induced by full length MAVS, whereas both proteins positively regulate cell death. To identify other polycistronic messages, we carried out genome-wide ribosomal profiling and identified a class of antiviral truncated variants. This study therefore reveals the existence of a functionally important bicistronic antiviral mRNA, and suggests a widespread role for polycistronic mRNAs in the innate immune system.

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6.

Inflammasomes are cytosolic multiprotein complexes that assemble in response to infectious or noxious stimuli and activate the CASPASE-1 protease. The inflammasome containing the nucleotide binding domainleucine-rich repeat (NBD-LRR) protein NLRC4 (interleukin-converting enzyme protease-activating factor [IPAF]) responds to the cytosolic presence of bacterial proteins such as flagellin or the inner rod component of bacterial type III secretion systems (e.g., Salmonella PrgJ). In some instances, such as infection with Legionella pneumophila, the activation of the NLRC4 inflammasome requires the presence of a second NBD-LRR protein, NAIP5. NAIP5 also is required for NLRC4 activation by the minimal C-terminal flagellin peptide, which is sufficient to activate NLRC4. However, NLRC4 activation is not always dependent upon NAIP5. In this report, we define the molecular requirements for NAIP5 in the activation of the NLRC4 inflammasome. We demonstrate that the N terminus of flagellin can relieve the requirement for NAIP5 during the activation of the NLRC4 inflammasome. We also demonstrate that NLRC4 responds to the Salmonella protein PrgJ independently of NAIP5. Our results indicate that NAIP5 regulates the apparent specificity of the NLRC4 inflammasome for distinct bacterial ligands.

Caspase-11–dependent cell death is controlled by carboxypeptidase B1 by inducing the cleavage of C3 and activation of C3aR.

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