m6A methylation potentiates cytosolic dsDNA recognition in a sequence-specific manner

Nucleic acid sensing through pattern recognition receptors is critical for immune recognition of microbial infections. Microbial DNA is frequently methylated at the N-6 position of adenines (m6A), a modification that is rare in mammalian host DNA. We show here how that m6A methylation of 5 '-GATC-3 ' motifs augments the immunogenicity of synthetic double-stranded (ds)DNA in murine macrophages and dendritic cells. Transfection with m6A-methylated DNA increased the expression of the activation markers CD69 and CD86, and of Ifn beta, iNos and Cxcl10 mRNA. Similar to unmethylated cytosolic dsDNA, recognition of m6A DNA occurs independently of TLR and RIG-I signalling, but requires the two key mediators of cytosolic DNA sensing, STING and cGAS. Intriguingly, the response to m6A DNA is sequence-specific. m6A is immunostimulatory in some motifs, but immunosuppressive in others, a feature that is conserved between mouse and human macrophages. In conclusion, epigenetic alterations of DNA depend on the context of the sequence and are differentially perceived by innate cells, a feature that could potentially be used for the design of immune-modulating therapeutics.

cGAS regulation and activity during genotoxic stress

Sélène-Véronique Glück

The innate immune system has evolved to detect pathogens through germlineencoded pattern recognition receptors (PRRs). Nucleic acid sensors, a defined class of PRRs, bind to microbial and viral nucleic acids and trigger the production of type I interferons and cytokines which mediate the first line of host defense. In the past, these sensors have been primarily studied in the context of infections. Yet, the DNA sensor cyclic GMP-AMP synthase (cGAS) has been recently described to detect not only microbial and viral derived dsDNA but also self-DNA in a sequenceindependent manner. At steady state, the organization of nuclear self-DNA is tightly controlled by a complex network of multiple factors that can become imbalanced during genotoxic stress. Whether and how this stress can be sensed by cGAS in the absence of infection are the main questions of this thesis. Observing the fate of cGAS in space and time in the presence of several genotoxic insults including chemotherapeutics allowed me to gain novel insights into processes regulating the localization and activation of cGAS. In this thesis, I demonstrate that cGAS localizes to both nuclear and cytosolic compartments of the cell. We show that cGAS is inactive in the nucleus due to binding to the acidic patch on H2A-H2B heterodimers. Upon genotoxic stress, cGAS is removed from the nucleus in a manner that is dependent on histones and remains inactive. Moreover, with time cGAS transcription is repressed resulting in a gradual decrease of cGAS levels. On the other hand, cGAS-activating DNA substrates such as cytosolic chromatin fragments accumulate in the cytosol of senescent cells deficient in Lamin B1 when cGAS levels are already decreased. Along with the occurrence of these chromatin fragments, cGAS mediates an inflammatory response dependent on STING (Stimulator of interferon genes), referred to as the senescence-associated secretory phenotype (SASP). Overall, we conclude that acute genotoxic stress does not result in cGAS-STING signaling, whereas the chronic DNA damage response, senescence, activates the cGAS-STING pathway. Moreover, the results presented in this work suggest a new role for cGAS-STING signaling during senescence beyond the recognition of pathogens.

EPFL2020

Induction of type I IFNs by intracellular DNA-sensing pathways

Andrea Ablasser

A successful antimicrobial immune response involves the coordinate action of cells and soluble factors, with the cytokine family of type I interferons (IFNs) having a central role. Type I IFNs are not only crucial in conferring immediate antimicrobial, most importantly antiviral effects, but they also have an essential role in bridging the innate with the adaptive immune response. Therefore, production of these key cytokines must be tightly controlled. To this effect the host has evolved a set of pattern recognition receptors (PRRs) that reliably and specifically detect the presence of microbial pathogens before mounting an IFN response. Most PRR pathways that are known to induce type I IFNs are triggered upon recognition of nucleic acids. This mode of sensing is not straightforward, as large amounts of RNA and DNA are also present within the host. Nevertheless, in some cases distinct molecular features that are present within foreign nucleic acids but absent in endogenous nucleic acids, allow the host to reliably discriminate between 'self' and 'non-self'. At the same time, compartmentalization of PRRs within subcellular organelles that are usually devoid of host nucleic acids, but are sites of pathogen localization, is another principle that enables the host to distinguish self from non-self. The latter mode of sensing applies to the detection of microbial DNA within the cytoplasm, a compartment in which host DNAs are usually not present. Despite the past years' tremendous progress in the field of innate immunity, our understanding of cytoplasmic DNA sensing mechanisms is only beginning to form/take form. In this review, we outline the recent advancements in the elucidation of intracellular DNA-sensing pathways and discuss the future directions of this emerging field.

2012

Exploiting immunofunctional nanoparticles as vaccines and synthetic pathogens

Sai Reddy

The emergence of recombinant DNA technology has shifted research towards developing subunit vaccines, which use protein or peptide antigens instead of live/attenuated pathogens. Recent research has also shown that the best way to get sustained immunity is to deliver an antigen directly to antigen-presenting cells (APCs), specifically dendritic cells (DCs). However there are two difficulties to overcome in targeting DCs for use in subunit vaccines: (1) DCs are present in a low concentration in peripheral tissue (where typical vaccines are delivered) compared to secondary lymphoid organs such as lymph nodes. Thus delivering antigen to DCs in lymph nodes offers a powerful alternative for vaccine technology. (2) Activating DCs requires co-delivering an adjuvant also sometimes known as a molecular "danger signal" which matures DCs and initiates the subsequent adaptive immune response. Adjuvants are needed since most protein and peptide antigens are insufficiently immunogenic. Current danger signals being explored in experimental vaccine formulations mimic bacterial components (e.g., lipopolysaccharide (LPS), unmethylated CpG DNA) and often signal through a class of pattern recognition receptors knows as Toll-like receptors (TLRs), however limitations exist in that these often cause toxic side effects by inducing high levels pro-inflammatory cytokines. To address the need to target DCs, we used a nanoparticle delivery approach. We investigated the delivery of 20, 45, and 100 nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consisted of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45 nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120 h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-containing DCs (and other APCs) was seen at 96 h vs. 24 h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45 nm showed potential for vaccine delivery specifically targeting DCs in lymph nodes. Next we exploited our nanoparticle technology as a vaccine platform. We engineered antigen-bearing nanoparticle vaccines with two novel features: lymph node-targeting and in situ complement activation. Following intradermal injection, ultra-small nanoparticles (25 nm) were readily transported by interstitial flows into lymphatic capillaries, which leads to their accumulation in lymph nodes resident DCs. We further designed the nanoparticle surface chemistry to activate the complement cascade, thus spontaneously generating a danger signal in situ for DC activation. With ovalbumin as a model antigen conjugated to the nanoparticles, we demonstrated humoral and cellular immunity in mice. Finally we exploited nanoparticles as "synthetic pathogens" to isolate the effect of free surface thiols on complement activation and the subsequent immunological response induced. We found that surfaces with both hydroxyl and thiol groups activated complement to significantly higher levels than hydroxl groups alone. We then demonstrated that nanoparticles that induced thiol-associated complement activation were potent danger signals that were able to mature dendritic cells to express high levels of costimulatory molecules and the Th1 promoting cytokine interuleukin-12. Moreover, this dendritic cell maturation was dependent on a Toll-like receptor 4 (TLR4) signaling pathway and independent of the adaptor protein myeloid differentiation protein 88 (MyD88). Thus we uncover a potential direct link between complement and TLR4 signaling that is dependent on the specific activation of complement by thiol-expressing surfaces. In conclusion, this thesis develops a novel vaccine methodology, which is at the interface of nanotechnology and biotechnology, by harnessing lymphatic transport to target DCs and complement activation to activate adaptive immunity; we demonstrated an elegant vaccine technology platform for applications that include global health. Finally we exploit our nanoparticles as synthetic pathogens to discover fundamental mechanisms connecting the complement system and TLR signaling.