Sarah Kim

Inflammasomes: current understanding and open questions

The innate immune system relies on its capability to detect invading microbes, tissue damage, or stress via evolutionarily conserved receptors. The nucleotide-binding domain leucine-rich repeat (NLR)-containing family of pattern recognition receptors includes several proteins that drive inflammation in response to a wide variety of molecular patterns. In particular, the NLRs that participate in the formation of a molecular scaffold termed the “inflammasome” have been intensively studied in past years. Inflammasome activation by multiple types of tissue damage or by pathogen-associated signatures results in the autocatalytic cleavage of caspase-1 and ultimately leads to the processing and thus secretion of pro-inflammatory cytokines, most importantly interleukin (IL)-1β and IL-18. Here, we review the current knowledge of mechanisms leading to the activation of inflammasomes. In particular, we focus on the controversial molecular mechanisms that regulate NLRP3 signaling and highlight recent advancements in DNA sensing by the inflammasome receptor AIM2.

Listeria monocytogenes is sensed by the NLRP3 and AIM2 Inflammasome

The inflammasome pathway functions to regulate caspase‐1 activation in response to a broad range of stimuli. Caspase‐1 activation is required for the maturation of the pivotal pro‐inflammatory cytokines of the pro‐IL‐1β family. In addition, caspase‐1 activation leads to a certain type of cell death known as pyroptosis. Activation of the inflammasome has been shown to play a critical role in the recognition and containment of various microbial pathogens, including the intracellularly replicating Listeria monocytogenes; however, the inflammasome pathways activated during L. monocytogenes infection are only poorly defined. Here, we demonstrate that L. monocytogenes activates both the NLRP3 and the AIM2 inflammasome, with a predominant involvement of the AIM2 inflammasome. In addition, L. monocytogenes‐triggered cell death was diminished in the absence of both AIM2 and NLRP3, and is concomitant with increased intracellular replication of L. monocytogenes. Altogether, these data establish a role for DNA sensing through the AIM2 inflammasome in the detection of intracellularly replicating bacteria.

Selection of molecular structure and delivery of RNA oligonucleotides to activate TLR7 versus TLR8 and to induce high amounts of IL-12p70 in primary human monocytes

Detection of non-self RNA by TLRs within endosomes and by retinoic acid-inducible gene I (RIG-I)-like helicases in the cytosol is central to mammalian antiviral immunity. In this study, we used pathway-specific agonists and targeted delivery to address RNA immunorecognition in primary human immune cells. Within PBMC, plasmacytoid dendritic cells (pDC) and monocytes were found to be responsible for IFN-α production upon immunorecognition of RNA. The mechanisms of RNA recognition in pDC and monocytes were distinct. In pDC, recognition of ssRNA and dsRNA oligonucleotides was TLR7-dependent, whereas a 5′ triphosphate moiety (RIG-I ligand activity) had no major contribution to IFN-α production. In monocytes, the response to RNA oligonucleotides was mediated by either TLR8 or RIG-I. TLR8 was responsible for IL-12 induction upon endosomal delivery of ssRNA oligonucleotides and RIG-I was responsible for IFN-α production upon delivery of 5′ triphosphate RNA into the cytosol. In conclusion, the dissection of these pathways by selecting the appropriate structure and delivery of RNA reveals pDC as major producer of IFN-α upon TLR-mediated stimulation and monocytes as major producer of IFN-α upon RIG-I-mediated stimulation. Furthermore, our results uncover the potential of monocytes to function as major producers of IL-12p70, a key Th1 cytokine classically ascribed to myeloid dendritic cells that cannot be induced by CpG oligonucleotides in the human system.

Characterizing the genetic basis of innate immune response in TLR4-activated human monocytes

Toll-like receptors (TLRs) play a key role in innate immunity. Apart from their function in host defense, dysregulation in TLR signalling can confer risk to autoimmune diseases, septic shock or cancer. Here we report genetic variants and transcripts that are active only during TLR signalling and contribute to interindividual differences in immune response. Comparing unstimulated versus TLR4-stimulated monocytes reveals 1,471 expression quantitative trait loci (eQTLs) that are unique to TLR4 stimulation. Among these we find functional SNPs for the expression of NEU4, CCL14, CBX3 and IRF5 on TLR4 activation. Furthermore, we show that SNPs conferring risk to primary biliary cirrhosis (PBC), inflammatory bowel disease (IBD) and celiac disease are immune response eQTLs for PDGFB and IL18R1. Thus, PDGFB and IL18R1 represent plausible candidates for studying the pathophysiology of these disorders in the context of TLR4 activation. In summary, this study presents novel insights into the genetic basis of the innate immune response and exemplifies the value of eQTL studies in the context of exogenous cell stimulation.

Self-priming determines high type I IFN production by plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are responsible for the robust and immediate production of type I IFNs during viral infection. pDCs employ TLR7 and TLR9 to detect RNA and CpG motifs present in microbial genomes. CpG‐A was the first synthetic stimulus available that induced large amounts of IFN‐α (type I IFN) in pDCs. CpG‐B, however, only weakly activates pDCs to produce IFN‐α. Here, we demonstrate that differences in the kinetics of TLR9 activation in human pDCs are essential for the understanding of the functional difference between CpG‐A and CpG‐B. While CpG‐B quickly induces IFN‐α production in pDCs, CpG‐A stimulation results in delayed yet maximal IFN‐α induction. Constitutive production of low levels of type I IFN in pDCs, acting in a paracrine and autocrine fashion, turned out to be the key mechanism responsible for this phenomenon. At high cell density, pDC‐derived, constitutive type I IFN production primes pDCs for maximal TLR responsiveness. This accounts for the high activity of higher structured TLR agonists that trigger type I IFN production in a delayed fashion. Altogether, these data demonstrate that high type I IFN production by pDCs cannot be simply ascribed to cell‐autonomous mechanisms, yet critically depends on the local immune context.

TLR8-driven IL-12-dependent reciprocal and synergistic activation of NK cells and monocytes by immunostimulatory RNA

Immunostimulatory RNA (isRNA) depending on sequence and structure can function as a ligand for Toll-like receptor (TLR) 7 and TLR8. Here we show that isRNA induces high levels of bioactive interleukin-12 in purified human monocytes, whereas purified natural killer (NK) cells did not respond. However, in a coculture of monocytes and NK cells, isRNA dramatically increased NK cell function. Activation of monocytes and NK cells was bidirectional, as monocytes in the presence of NK cells produced higher levels of bioactive interleukin-12. As a result of the monocyte-NK cell interaction in peripheral blood mononuclear cells isRNA induced high levels of interferon (IFN)-γ in NK cells and strong NK cell-mediated cytotoxic activity. Induction of simultaneous IFN-γ production and lytic activity by isRNA in NK cells was higher as compared with other established nucleic acid or small molecule TLR ligands. Our studies demonstrate that monocytes play a pivotal role in the orchestration of a strong NK cell response. With early NK cell-dependent IFN-γ production being critical for the development of antigen-specific cytotoxic T lymphocyte responses, newly developed isRNA-based TLR8 ligands join the list of promising oligonucleotides for immunotherapy of viral infection and cancer.

Whole exome sequencing and array-based molecular karyotyping as aids to prenatal diagnosis in fetuses with suspected Simpson-Golabi-Behmel syndrome

Simpson–Golabi–Behmel (SGBS) syndrome type 1 and type 2 represent rare X‐linked prenatal overgrowth disorders. The aim of our study is to describe the prenatal sonographic features as well as the genetic work‐up.

De novo nonsense and frameshift variants of TCF20 in individuals with intellectual disability and postnatal overgrowth.

Recently, germline variants of the transcriptional co-regulator gene TCF20 have been implicated in the aetiology of autism spectrum disorders (ASD). However, the knowledge about the associated clinical picture remains fragmentary. In this study, two individuals with de novo TCF20 sequence variants were identified in a cohort of 313 individuals with intellectual disability of unknown aetiology, which was analysed by whole exome sequencing using a child–parent trio design. Both detected variants – one nonsense and one frameshift variant – were truncating. A comprehensive clinical characterisation of the patients yielded mild intellectual disability, postnatal tall stature and macrocephaly, obesity and muscular hypotonia as common clinical signs while ASD was only present in one proband. The present report begins to establish the clinical picture of individuals with de novo nonsense and frameshift variants of TCF20 which includes features such as proportionate overgrowth and muscular hypotonia. Furthermore, intellectual disability/developmental delay seems to be fully penetrant amongst known individuals with de novo nonsense and frameshift variants of TCF20, whereas ASD is shown to be incompletely penetrant. The transcriptional co-regulator gene TCF20 is hereby added to the growing number of genes implicated in the aetiology of both ASD and intellectual disability. Furthermore, such de novo variants of TCF20 may represent a novel differential diagnosis in the overgrowth syndrome spectrum.

An unexpected role for RNA in the recognition of DNA by the innate immune system

A central function of our innate immune system is to sense microbial pathogens by the presence of their nucleic acid genomes or their transcriptional or replicative activity. In mammals, a receptor-‐based system is mainly responsible for the detection of these “non-‐ self” nucleic acids. Tremendous progress has been made in the past years to identify host constituents that are required for this intricate task. With regard to the sensing of RNA genome based pathogens by our innate immune system, a picture is emerging that includes certain families of the toll-‐like receptor family (TLR3, TLR7, TLR8) and the RIG-‐ I like helicases (RIG-‐I, MDA5 and LGP2). Genetic loss of function studies implicate that the absence of these pathways can lead to a complete lack of recognition of certain RNA viruses. At the same time, intracellular DNA can also trigger potent innate immune responses, yet the players in this field are less clear. We and another group recently identified a role for RNA polymerase III in the conversion of AT-‐rich DNA into an RNA ligand that is sensed by the RIG-‐I pathway. In this review article, we will discuss the mechanistics and implications of this novel pathway.