Jonathan C. Kagan

TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta.

Toll-like receptor 4 (TLR4) induces two distinct signaling pathways controlled by the TIRAP-MyD88 and TRAM-TRIF pairs of adaptor proteins, which elicit the production of proinflammatory cytokines and type I interferons, respectively. How TLR4 coordinates the activation of these two pathways is unknown. Here we show that TLR4 activated these two signaling pathways sequentially in a process organized around endocytosis of the TLR4 complex. We propose that TLR4 first induces TIRAP-MyD88 signaling at the plasma membrane and is then endocytosed and activates TRAM-TRIF signaling from early endosomes. Our data emphasize a unifying theme in innate immune recognition whereby all type I interferon–inducing receptors signal from an intracellular location.

Phosphoinositide-Mediated Adaptor Recruitment Controls Toll-like Receptor Signaling

Toll-like receptors (TLRs) play a critical role in the immune system as sensors of microbial infection. Signaling downstream from TLRs is initiated by the recruitment of adaptor proteins, including MyD88 and TIRAP. These adaptors play essential roles in TLR signaling, but the mechanism of their function is currently unknown. Here we demonstrate that TIRAP and MyD88 have distinct functions and describe a mechanism of recruitment of TIRAP and MyD88 to TLR4. We find that TIRAP contains a phosphatidylinositol 4,5-bisphosphate (PIP2) binding domain, which mediates TIRAP recruitment to the plasma membrane. TIRAP then functions to facilitate MyD88 delivery to activated TLR4 to initiate signal transduction. These results establish that phosphoinositide-mediated adaptor recruitment initiates a specific signal-transduction pathway.

A cell biological view of Toll-like receptor function: regulation through compartmentalization

An emerging paradigm in innate immune signalling is that cell biological context can influence the outcome of a ligand–receptor interaction. In this Review we discuss how Toll-like receptor (TLR) activation and signal transduction are regulated by subcellular compartmentalization of receptors and downstream signalling components. In particular, we focus on the functional specialization of TLRs in the endosomal system. We discuss recent studies that illustrate how basic aspects of the cellular machinery contribute to TLR function and regulation. This emerging area of research will provide important information on how immune signal transduction networks depend on (and in some cases influence) the generic regulators that organize eukaryotic cells.

Peroxisomes are signaling platforms for antiviral innate immunity.

Peroxisomes have long been established to play a central role in regulating various metabolic activities in mammalian cells. These organelles act in concert with mitochondria to control the metabolism of lipids and reactive oxygen species. However, while mitochondria have emerged as an important site of antiviral signal transduction, a role for peroxisomes in immune defense is unknown. Here, we report that the RIG-I-like receptor (RLR) adaptor protein MAVS is located on peroxisomes and mitochondria. We find that peroxisomal and mitochondrial MAVS act sequentially to create an antiviral cellular state. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, which amplifies and stabilizes the antiviral response. The interferon regulatory factor IRF1 plays a crucial role in regulating MAVS-dependent signaling from peroxisomes. These results establish that peroxisomes are an important site of antiviral signal transduction.

CD14 Controls the LPS-Induced Endocytosis of Toll-like Receptor 4

The transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLCγ2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.

Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites

It is unknown how Legionella pneumophila cells escape the degradative lysosomal pathway after phagocytosis by macrophages and replicate in an organelle derived from the endoplasmic reticulum. Here we show that, after internalization, L. pneumophila-containing phagosomes recruit early secretory vesicles. Once L. pneumophila phagosomes have intercepted early secretory vesicles they begin to acquire proteins residing in transitional and rough endoplasmic reticulum. The functions of Sar1 and ADP-ribosylation factor-1 are important for biogenesis of the L. pneumophila replicative organelle. These data indicate that L. pneumophila intercepts vesicular traffic from endoplasmic-reticulum exit sites to create an organelle that permits intracellular replication and prevents destruction by the host cell.

Innate Immune Pattern Recognition: A Cell Biological Perspective

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.

Legionella Subvert the Functions of Rab1 and Sec22b to Create a Replicative Organelle

Legionella pneumophila is a bacterial pathogen that infects eukaryotic host cells and replicates inside a specialized organelle that is morphologically similar to the endoplasmic reticulum (ER). To better understand the molecular mechanisms governing transport of the Legionella-containing vacuole (LCV), we have identified host proteins that participate in the conversion of the LCV into a replicative organelle. Our data show that Rab1 is recruited to the LCV within minutes of uptake. Rab1 recruitment to the LCV precedes remodeling of this compartment by ER-derived vesicles. Genetic inhibition studies demonstrate that Rab1 is important for the recruitment of ER-derived vesicles to the LCV and that inhibiting Rab1 function abrogates intracellular growth of Legionella. Morphological studies indicate that the Sec22b protein is located on ER-derived vesicles recruited to the LCV and that Sec22b is delivered to the LCV membrane. Sec22b function was found to be important for biogenesis of the specialized organelle that supports Legionella replication. These studies demonstrate that Legionella has the ability to subvert Rab1 and Sec22b function to facilitate the transport and fusion of ER-derived vesicles with the LCV, resulting in the formation of a specialized organelle that can support bacterial replication.

Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth

Legionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV‐related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein–protein interactions. Specific interactions between different Icm proteins were detected by yeast two‐hybrid and gel overlay analysis. These data support a model in which the IcmQ–IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS–IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.

A Promiscuous Lipid-Binding Protein Diversifies the Subcellular Sites of Toll-like Receptor Signal Transduction

The Toll-like receptors (TLRs) of the innate immune system are unusual in that individual family members are located on different organelles, yet most activate a common signaling pathway important for host defense. It remains unclear how this common signaling pathway can be activated from multiple subcellular locations. Here, we report that, in response to natural activators of innate immunity, the sorting adaptor TIRAP regulates TLR signaling from the plasma membrane and endosomes. TLR signaling from both locations triggers the TIRAP-dependent assembly of the myddosome, a protein complex that controls proinflammatory cytokine expression. The actions of TIRAP depend on the promiscuity of its phosphoinositide-binding domain. Different lipid targets of this domain direct TIRAP to different organelles, allowing it to survey multiple compartments for the presence of activated TLRs. These data establish how promiscuity, rather than specificity, can be a beneficial means of diversifying the subcellular sites of innate immune signal transduction.