Nucleic acid recognition by the innate immune system.

Receptors of the innate immune system recognize conserved microbial features and provide key signals that initiate immune responses. Multiple transmembrane and cytosolic receptors have evolved to recognize RNA and DNA, including members of the Toll-like receptor and RIG-I-like receptor families and several DNA sensors. This strategy enables recognition of a broad range of pathogens; however, in some cases, this benefit is weighed against the cost of potential self recognition. Recognition of self nucleic acids by the innate immune system contributes to the pathology associated with several autoimmune or autoinflammatory diseases. In this review, we highlight our current understanding of nucleic acid sensing by innate immune receptors and discuss the regulatory mechanisms that normally prevent inappropriate responses to self.

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.

Toll-like receptor 2 on inflammatory monocytes induces type I interferon in response to viral but not bacterial ligands.

Despite the paradigm that the innate immune system uses nucleic acid-specific receptors to detect viruses because of a lack of other conserved features, many viruses are recognized by Toll-like receptor 2 (TLR2) and TLR4. The relevance of this recognition for antiviral immunity remains largely unexplained. Here we report that TLR2 activation by viruses led to the production of type I interferon. TLR2-dependent induction of type I interferon occurred only in response to viral ligands, which indicates that TLR2 is able to discriminate between pathogen classes. We demonstrate that this specialized response was mediated by Ly6C(hi) inflammatory monocytes. Thus, the innate immune system can detect certain non-nucleic acid features of viruses and links this recognition to the induction of specific antiviral genes.

Transmembrane mutations in Toll-like receptor 9 bypass the requirement for ectodomain proteolysis and induce fatal inflammation.

Recognition of nucleic acids as a signature of infection by Toll-like receptors (TLRs) 7 and 9 exposes the host to potential self-recognition and autoimmunity. It has been proposed that intracellular compartmentalization is largely responsible for reliable self versus nonself discrimination by these receptors. We have previously shown that TLR9 and TLR7 require processing prior to activation, which may further reinforce receptor compartmentalization and tolerance to self, yet this possibility remains untested. Here we report that residues within the TLR9 transmembrane (TM) region conferred the requirement for ectodomain proteolysis. TLR9 TM mutants responded to extracellular DNA, and mice expressing such receptors died from systemic inflammation and anemia. This inflammatory disease did not require lymphocytes and appeared to require recognition of self-DNA by dendritic cells. To our knowledge, these results provide the first demonstration that TLR-intrinsic mutations can lead to a break in tolerance.

Local TNFR1 Signaling Licenses Murine Neutrophils for Increased TLR-Dependent Cytokine and Eicosanoid Production.

Neutrophils are generally the first immune cells recruited during the development of sterile or microbial inflammation. As these cells express many innate immune receptors with the potential to directly recognize microbial or endogenous signals, we set out to assess whether their functions are locally influenced by the signals present at the onset of inflammation. Using a mouse model of peritonitis, we demonstrate that neutrophils elicited in the presence of C-type lectin receptor ligands have an increased ability to produce cytokines, chemokines, and lipid mediators in response to subsequent TLR stimulation. Importantly, we found that licensing of cytokine production was mediated by paracrine TNF-α-TNFR1 signaling rather than direct ligand sensing, suggesting a form of quorum sensing among neutrophils. Mechanistically, licensing was largely imparted by changes in the posttranscriptional regulation of inflammatory cytokines, whereas production of IL-10 was regulated at the transcriptional level. Altogether, our data suggest that neutrophils rapidly adapt their functions to the local inflammatory milieu. These phenotypic changes may promote rapid neutrophil recruitment in the presence of pathogens but limit inflammation in their absence.

Diversity-generating retroelement homing regenerates target sequences for repeated rounds of codon rewriting and protein diversification.

Diversity-generating retroelements (DGRs) introduce vast amounts of sequence diversity into target genes. During mutagenic homing, adenine residues are converted to random nucleotides in a unidirectional, reverse transcriptase-dependent transposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using a Bordetella bacteriophage DGR as a model, we demonstrate that homing occurs through a TR-containing RNA intermediate and is RecA independent. Marker transfer studies show that cDNA integration at the 3' end of VR occurs within a (G/C)(14) element, and deletion analysis demonstrates that the reaction is independent of 5' end cDNA integration. cDNA integration at the 5' end of VR requires only short stretches of sequence homology. We propose that homing occurs through a unique target DNA-primed reverse transcription mechanism that precisely regenerates target sequences. This nonproliferative "copy and replace" mechanism enables repeated rounds of protein diversification and optimization of ligand-receptor interactions.

Selective ligand recognition by a diversity-generating retroelement variable protein.

Diversity-generating retroelements (DGRs) recognize novel ligands through massive protein sequence variation, a property shared uniquely with the adaptive immune response. Little is known about how recognition is achieved by DGR variable proteins. Here, we present the structure of the Bordetella bacteriophage DGR variable protein major tropism determinant (Mtd) bound to the receptor pertactin, revealing remarkable adaptability in the static binding sites of Mtd. Despite large dissimilarities in ligand binding mode, principles underlying selective recognition were strikingly conserved between Mtd and immunoreceptors. Central to this was the differential amplification of binding strengths by avidity (i.e., multivalency), which not only relaxed the demand for optimal complementarity between Mtd and pertactin but also enhanced distinctions among binding events to provide selectivity. A quantitatively similar balance between complementarity and avidity was observed for Bordetella bacteriophage DGR as occurs in the immune system, suggesting that variable repertoires operate under a narrow set of conditions to recognize novel ligands.

MicroRNAs and LPS: developing a relationship in the neonatal gut.

Upon birth, the intestine converts from a sterile environment to a home for commensal microorganisms. How immune homeostasis is maintained during this transition is not well understood. Here, Chassin et al. (2010) demonstrate that microRNA-146a regulates the responsiveness of intestinal epithelial cells during microbial colonization of the neonatal intestine.

A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP.

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di-guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor kappaB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid-sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.