Elsevier

Methods in Enzymology

Volume 625, 2019, Pages 269-285
Methods in Enzymology

Chapter Sixteen - Assessment of inflammasome and type I IFN responses to DNA viruses and DNA PAMPS

https://doi.org/10.1016/bs.mie.2019.05.008Get rights and content

Abstract

The innate immune system is an evolutionarily conserved host defense system and is the first barrier to infection. The system utilizes genetically conserved receptors to identify the presence of microbial structures. Engagement of innate immune receptors by primarily by ligands that discriminate pathogens from the host activates programmed responses that limit pathogen expansion. Despite its ubiquitous nature, surprisingly DNA is a critical structure that triggers innate immune responses. Focusing on structural modifications or aberrant location of DNA, innate immune receptors identify physiologic stress. Inflammasomes and interferons are critical innate immune pathways that are activated by DNA. DNA binding proteins that tie recognition of DNA to both programmed responses have been identified, and their importance demonstrated in infection models. In this chapter, we discuss techniques to analyze AIM2 inflammasome and cGAS interferon activation by synthetic DNA and DNA viruses. We also discuss methods to measure the activity of these immune pathways.

Introduction

The innate immune system is an ancient evolutionarily conserved host defense system (Buchmann, 2014). The innate immune system in humans is comprised of several haematopoietically derived cells that perform specialized functions. However, virtually all cells have intrinsic immune defenses which act as an early warning system while limiting pathogen spread. The combined actions of these specialized immune cells and tissue resident cells serve as the first line of defense against invading microbial pathogens and other toxic insults.

Activating an innate immune response involves complex interactions between the pathogen and the host. Membrane-localized, endosomal, cytosolic and nuclear germ-line encoded sensors, collectively referred to as pattern recognition receptors (PRR) are critical to recognizing the presence of pathogens and initiating innate immune responses. Innate immune PRRs, prototypically Toll-like receptors (TLR)s (Kawai & Akira, 2010), expressed by tissue-resident or hematopoietic cells, “sense” pathogen-associated molecular patterns (PAMPs) and trigger downstream effector programs. Besides TLRs, other innate PRRs include (1) Nucleotide-binding Oligomerization Domain (NOD)-like receptors, (2) complement receptors, (3) scavenger receptors (e.g., CD36, Scavenger Receptor [SR]-A, Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 [LOX-1], and Drosophila SR-C [dSRsingle bondC]), (4) C-type lectin receptors (e.g., mannose-binding proteins, dectin-1, dectin-2, and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin [DC-SIGN]), and (5) intracellular nucleic acid-sensing receptors (e.g., Absent in Melanoma 2 [AIM2], Melanoma Differentiation-Associated protein 5 [MDA-5], Retinoic-Acid Inducible Gene I [RIG-I], and cyclic GMP-AMP synthetase [cGAS]). Triggering of a PRR activates programmed responses designed to limit the expansion of the pathogen and support the development of adaptive immunity and immunologic memory. Inflammasomes and interferon type I (IFN-I) are examples of important programmed responses with antimicrobial effects.

The recognition or “sensing” of nucleic acids is a primary mechanism used by the innate immune system to mount an immune response against microbial pathogens. dsRNA and ssRNA can be recognized in the endosomal system by some TLRs leading to the production of IFN-I and other inflammatory cytokines (Kawai & Akira, 2010). In the cytoplasm, dsRNA is recognized by MDA5 and RIG-I; the latter also sensing 5′ triphosphate RNA. Binding of these RNA species to MDA5 and RIG-I recruits Mitochondrial antiviral-signaling protein (MAVS) leading to downstream activation of IRF3, MAPK, and NF-κB (Kato, Takahasi, & Fujita, 2011).

Like RNA, the presence of dsDNA can also be recognized in the endosome by TLRs leading to production on IFN-I. Several cytosolic DNA sensors including Gamma-interferon-inducible protein-16 (IFI16) and DEAD-Box Helicase 41 (DDX41) have been described. However, the mechanism by which these receptors mediate a DNA-dependent immune response is not entirely understood (Unterholzner et al., 2010; Zhang et al., 2011). Recently, it was recognized that cGAS is a significant driver of IFN-I expression in response to dsDNA in the cytoplasm (Sun, Wu, Du, Chen, & Chen, 2013; Wu et al., 2013). Recognition of dsDNA by cGAS induces enzymatic conversion of ATP and GTP into the second messenger, cyclic di-GMP-AMP (cGAMP) (Gao et al., 2013; Li et al., 2013). cGAMP then binds to Stimulator of interferon genes (STING), also known as transmembrane protein 173 (TMEM173) which resides in the endoplasmic reticulum. cGAMP binding to STING dimers induces a conformational change, and STING then binds to TANK-Binding Kinase 1 (TBK1) and translocates to the perinuclear Golgi region (Ishikawa, Ma, & Barber, 2009). TBK1 phosphorylates STING, allowing the recruitment of IRF3 which, after binding to STING, gets phosphorylated and activated by TBK1 (Liu et al., 2015). Binding of DNA and activation of cGAS is sequence-independent, and a broad spectrum of viral/bacterial pathogens, as well as endogenous DNA, induce IFN-I responses through the cGAS-STING-IRF3 axis. There is emerging evidence from monogenic interferonopathies and related mouse models that DNA sensing by the cGAS-STING pathway may be involved in the pathogenesis of autoinflammatory disorders (Crow & Manel, 2015).

Infectious diseases as a result of DNA virus infections are a significant health concern worldwide, and a thorough mechanistic understanding of host anti-viral responses is central to the development of antivirals and vaccines. Type I interferon production by the host is the frontline anti-viral defense strategy, and it is one of the primary immune responses to DNA viruses.

As previously discussed, IFN-I responses as a result of sensing viral DNA can be initiated in the cytoplasm and endosomal compartments. Many DNA virus are detected in endosomes by TLR9 and to a lesser extent TLR7 (Cerullo et al., 2007; Delale et al., 2005; Krug et al., 2004; Rasmussen et al., 2007; Tabeta et al., 2004). Most DNA viruses access the cytosolic compartment during their life cycle providing an opportunity to the innate immune system for early detection and response. Therefore, the detection of viral products in the cytosol is integral to innate anti-viral defense. One of the most commonly observed virus-associated molecular patterns in the cytosol is viral nucleic acid the detection of which forms the basis of the cytosolic sensing of DNA viruses. Several DNA viruses including herpes simplex virus 1, hepatitis B virus, vaccinia virus, adenovirus, and Kaposi's sarcoma-associated herpesvirus are recognized by the cGAS (Dansako et al., 2016; Lam, Stein, & Falck-Pedersen, 2014; Wu et al., 2015). Additional receptors, such as DAI, RNA polymerase III, IFI16 and others, have been implicated in recognition of DNA after viral infection (Chiu, Macmillan, & Chen, 2009; DeFilippis, Alvarado, Sali, Rothenburg, & Fruh, 2010; Unterholzner et al., 2010). These receptors appear to utilize a common signaling pathway which converges on STING to turn on interferon production at the transcriptional level.

Recognition of bacterial DNA is also used as a mechanism to mount responses to bacteria rapidly. Infection with Mycobacterium tuberculosis, Listeria monocytogenes, Chlamydia trachomatis, and Francisella tularensis activates IFN-I expression that is dependent on cGAS activity and bacteria-produced cyclic dinucleotides that act directly on STING (Collins et al., 2015; Hansen et al., 2014; Storek, Gertsvolf, Ohlson, & Monack, 2015; Watson et al., 2015; Woodward, Iavarone, & Portnoy, 2010).

Inflammasomes are macromolecular platforms for the recruitment and activation of inflammatory caspases in the context of stress or danger signals. Inflammasome complexes are composed of (1) a PRR, (2) the adaptor molecule, apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain) (ASC) and (3) the protease pro-caspase-1. Five inflammasomes have been identified in humans; they are named by their core PRR and include (1) the Nod-like receptor (NLR)s (e.g., NLRP1, NLRP3, NLRC4), (2) the PYHIN Pyrin and HIN domain (PYHIN) molecule AIM2 and (3) a third group comprised of the tripartite motif family member pyrin. Regulation of inflammation through the control of IL-1β and IL-18 production appears to be a major function of inflammasomes. IL-1β is a major pro-inflammatory cytokine that initiates inflammatory cascades and mediates immune-cell recruitment and activation (Netea, van de Veerdonk, van der Meer, Dinarello, & Joosten, 2015). IL-18 is a multifunctional cytokine that exerts many context-dependent biological effects including induction of IFN-γ and perforin from NK and T-cells, activation of mast cells and basophils and promoting tissue remodeling and wound healing (Samarani et al., 2016).

Assembly and activation of inflammasomes are tightly regulated and is induced by two signals (Chen & Ichinohe, 2015; He, Hara, & Nunez, 2016). Signal 1 or priming induces deubiquitination and dephosphorylation of NLRP3 as well as the transcription of inflammasome components and pro-cytokines. Signal 1 is mediated by the transcription factor NFκB and can be initiated by intracellular and plasma membrane PRR including TLR, cytokine receptors as well as other NFκB activators. Signal 2 or activation is less defined but involves potassium efflux, calcium mobilization from intracellular store compartments, and the production of ROS leads to assembly of the inflammasome complex. NACHT, LRR and PYD domains-containing protein 3 (NLRP3) is the most studied member of the NLR family. NLRP3 is activated in innate immune cells by a variety of stimuli, including pathogens and danger signals such as monosodium urate and ATP (Yao et al., 2017). Upon stimulation, NLRP3 undergoes conformational changes allowing recruitment and polymerization of the adaptor ASC—which is visualized as an approx. 1 μm speck in the cytoplasm—through PYD–PYD domain association. ASC filaments recruit pro-caspase-1 through CARD–CARD domain interactions to form the signaling complex. Autocleavage and the removal of regulatory domains generate the tetrameric activated caspase-1 which cleaves pro-IL-1β and pro-IL-18 to form mature, active IL-1β and IL-18 (Coll, O'Neill, & Schroder, 2016; Guo, Callaway, & Ting, 2015). Inflammasomes may also mediate a form of cell death called pyroptosis through cleavage of Gasdermin-D which can form pores in the plasma membrane (He et al., 2016).

Diverse microbes activate NLRP3 inflammasomes and mice lacking NLRP3, ASC, or caspase-1 are more susceptible to infection with many bacteria (Ceballos-Olvera, Sahoo, Miller, Del Barrio, & Re, 2011; Costa et al., 2012). The NLRP3 inflammasome is also activated by a number of RNA viruses underscoring the critical role of this innate immune mechanism in viral detection and defense. Prominent human pathogens that are recognized by NLRP3 include Influenza virus, respiratory syncytial virus, hepatitis C virus and HIV (Chattergoon et al., 2014; Kanneganti et al., 2006; Veenhuis et al., 2017). NLRP3 also recognizes enveloped and nonenveloped DNA viruses, such as herpes viruses, MVA and adenovirus (Delaloye et al., 2009; Muruve et al., 2008; Nour et al., 2011). Dysregulated NLRP3 activity is observed with a variety of inflammatory disorders, such as cryopyrin-associated periodic syndromes and contributes to the pathogenesis of metabolic diseases including diabetes (Masters et al., 2010). Of note sustained IL-18 elevation is also a hallmark of HIV-1 and HCV infections but clear mechanistic linkages between NLPR3 and increased rates of coronary artery disease, diabetes, etc. are yet to be established in this setting (Ahmad, Sindhu, Toma, Morisset, & Ahmad, 2002; Chattergoon et al., 2011; Torre et al., 2000; Veenhuis et al., 2017). While recent advances have been made in understanding inflammasome activation, NLPR3 regulation is not well understood and significant ambiguities about activation and inhibition of inflammasome-complex formation remain.

AIM2 is also a key sensor of pathogens that detects the presence of foreign DNA accumulating in the cytosol during the life cycle of intracellular pathogens including viruses, bacteria, and parasites. AIM2 achieves specificity by identifying altered or mislocalized DNA molecules. It can detect damaged DNA and the aberrant presence of DNA within the cytosolic compartment such as genomic DNA released into the cytosol upon loss of nuclear envelope integrity. dsDNA binds to AIM2 (absent in melanoma 2) in the cytoplasm, driving the activation of the inflammasome, the expression of IL-1β and IL-18, and the activation of pyroptosis in a caspase-1-dependent manner (Fernandes-Alnemri et al., 2010; Hornung et al., 2009). AIM2 appears to be able to recognize DNA molecules independently of the sequence and compared to other DNA sensors AIM2 is specialized in inflammasome assembly rather than promoting type I IFN responses.

Transfection of host, bacterial and viral DNA, besides its effects on the transcriptional activation of type-I IFN, has been shown to initiate a cascade leading to the proteolytic maturation of IL-1β. These DNA molecules relied on an ASC-dependent, NLRP3-independent but AIM2 dependent inflammasome (Muruve et al., 2008). During infections with intracellular pathogens, microbial DNA can be released in the cytosol. AIM2 function as a DNA sensor has been extensively described in the context of several microbes and pathogens; many bacterial species have been found to activate AIM2 including Francisella tularensis, Listeria monocytogenes, Streptococcus pneumoniae, species of Mycobacterium as well as Legionella pneumophila and Staphylococcus aureus. These observations have been extensively reviewed recently (Lugrin & Martinon, 2017).

Cytomegalovirus (CMV), Herpes Simplex Virus 1 (HSV-1), vaccinia virus, and human papillomavirus have been shown to induce AIM2 inflammasome (Hornung et al., 2009; Huang et al., 2017; Man et al., 2015; Rathinam et al., 2010; Reinholz et al., 2013). The mechanism by which viral DNA is liberated and exposed to AIM2 in the cytosol is not clearly defined. Viruses have been shown to evade AIM2 sensing possibly by inhibiting the ability of AIM2 to interact with released DNA. A recent study reported that Epstein–Barr virus (EBV) may activate AIM2 in THP1 cells (Torii et al., 2017). However, despite that AIM2 can detect viral DNA its contribution to viral immunity is still unclear.

Section snippets

Protocols

In this chapter, we will focus on the induction of type 1 interferons and inflammasomes in human immune cells. The reader should be aware that methods to activate similar pathways in murine cells and non-immune cells in humans differ slightly. The THP-1 cell line is extensively used in studies of innate immunity. When undifferentiated, this cell line mimic human monocytes. Several agents can be used to trigger differentiation to a macrophage phenotype. In this state, type I interferons and

Measurement of interferon type I (ELISA, qPCR, luciferase)

Note: Measurement of IFN-I activity can be done through commercial ELISAs, qPCR gene expression assays, and cell-based reporter luciferase assays.

Measurement of IFN-α by ELISA

Materials

  1. 1.

    Human Interferon alpha-1 SimpleStep ELISA® Kit (Abcam)

  2. 2.

    Spectrophotometer

Protocol
  1. We follow the manufacturer's recommendations which are outlined below with the following modifications.

  • 1.

    Prepare the appropriate amount of antibody coated well strips. We typically perform measurements on each sample in duplicate or triplicate.

  • 2.

    Equilibrate all reagents, experimental

Summary

Inflammasomes are key players in the regulation of cellular stress and immune responses. The PAMP ligands of certain key inflammasome receptors, such as NLPR3 are still poorly characterized. In contrast, AIM2 recognizes and binds to microbial DNA allowing it to function as a PRR and activate inflammasomes. Recent studies have demonstrated that AIM2 detects mislocalized self-DNA thereby allowing it to sense cellular perturbations leading to loss of cell viability and disruption of nuclear

Acknowledgment

M.A.C. is supported by supported by a grants AI102696 NIH/NIAID and P30 AI094189-01A1 NIH/NIAID.

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