Is inflammation a direct response to dsDNA breaks?

Mutat Res Fund Mol Mech Mutagen 808 (2018) 48–52

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Mutat Res Fund Mol Mech Mutagen journal homepage: www.elsevier.com/locate/mut

Is inflammation a direct response to dsDNA breaks? ⁎

T

Shahid Chaudhary, Gorantla Venkata Raghuram, Indraneel Mittra

Translational Research Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi-Mumbai, 410210, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Cell-free chromatin DNA damage dsDNA breaks Inflammation NFκB

Recent research shows that extra-nuclear cell-free chromatin (cfCh) released from dying cells can freely enter into healthy cells and integrate into their genomes. Genomic integration of cfCh leads to dsDNA breaks and activation of inflammatory cytokines both of which occur concurrently with similar kinetics and that induction of inflammation can be abrogated by preventing DNA breaks with the use of cfCh inactivating agents. The proposal is put forward that inflammatory cytokines are a new family of DDR proteins that are activated following dsDNA breaks inflicted by genomic integration of cfCh.

The human body is constantly challenged by insults such as tissue injury, diverse toxins and microorganisms, cosmic and medical quality radiations and air pollutants. The initial defence mechanism of the body is acute inflammation instigated by innate immune cells viz., peripheral polymorphonuclear neutrophils, eosinophils and macrophages [1]. Among other responses, the activated immune cells react by producing a spectrum of endogenous reactive oxygen species (ROS) [2]. The predominant genotoxic effect of ROS is DNA base or sugar damage that generally lead to single-strand DNA breaks [3]. ROS is also the main protagonist in the genesis of an inflammatory response [4]. However, ROS and NFκB share a complex relationship and the molecular mechanisms responsible for induction of NFκB by ROS remains largely obscure. ROS can have both stimulatory and inhibitory roles in NFκB signalling; while on the other hand, certain NFκB-dependent genes apparently regulate ROS production in the cell [4,5]. Furthermore, activation of poly (ADP-ribose) polymerase (PARP) and that of the tumour suppressor gene BRCA1, both of which are involved in DNA repair, leads to up-regulation of NFκB dependant gene transcription and immune-related genes such as IFN-γ [6,7]. Two recent articles describe a new class of endogenous DNA damaging agents in the form of extracellular nucleic acids which predominantly cause dsDNA breaks [8,9]. Initially, extracellular nucleic acids were considered as cellular junk but are now been increasingly subjected to scientific scrutiny [10]. The DNA-damaging nucleic acids that were examined in the first study cited above [8] comprised of cellfree DNA (cfDNA) and cell-free chromatin (cfCh) isolated from sera of healthy individuals and cancer patients. These are derived from the billions of cells that die in the body everyday and are found both in circulation and in other extracellular compartments of the body. Although, cfCh exists in circulation in its native form, it is unclear as to

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whether naked cfDNA exists as such or is derived from cfCh as a result of DNA extraction procedure. This study reported for the first time that cfDNA and cfCh can freely enter into recipient cells, such as mouse fibroblasts, and that their uptake is dependent on active cellular metabolism. Although fluorescently labeled cfDNA and cfCh were avidly taken up by the recipient cell nuclei reaching a maximum at 30 min and 6 h repectively, they were rapidly degraded such that only about 5% of nuclei showed fluorescent signals by 48 h (8). The intracellular cfDNA and cfCh associated themselves with host cell chromosomes triggering a robust DNA-damage-repair-response (DDR) with activation of multiple proteins of the DDR pathway. cfCh was consistently more active in inducing DDR than was cfDNA [8]. The activation of DDR by intracellular cfCh led to their genomic integration, and tens of thousands of human sequence reads were discovered on whole genome sequencing as was the detection of multiple human Alu sequences in the recipient mouse cells [8]. Although these findings did not conclusively prove that the incoming cfCh had integrated into the host cell genomes but remained episomal, the finding that FISH could detect presence of human DNA signals in single cell clones develop from cfCh treated mouse cells strongly suggested DNA integration. Nonetheless, long read sequencing technology which allows identification of novel junctions and sites of integration of human DNA in mouse genomes would be required for conclusive proof. The proposed mechanism of genomic integration of cfCh involves a model in which DDR plays a central role [8]. Classically, activation of DDR follows damage to DNA by agents such as ionizing and UV radiation, chemicals, free radicals, etc. which then attempts to repair the damage. In the proposed model, the uptaken intracellular cfCh deceives the cell into perceiving them as broken pieces of “self DNA” leading to activation of DDR prior to any damage to DNA having occurred. The

Corresponding author. E-mail address: [email protected] (I. Mittra).

https://doi.org/10.1016/j.mrfmmm.2018.02.002 Received 23 October 2017; Received in revised form 13 January 2018; Accepted 13 February 2018 Available online 22 February 2018 0027-5107/ © 2018 Elsevier B.V. All rights reserved.


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Fig. 1. Schematic representation of proposed model of DNA damage and inflammation. NHEJ = non-homologous end-joining; HR = homologous recombination; NHR = non-homologous recombination. Reproduced with modification from J. Biosci 2015; 40: 91–111.

inflammatory cytokine IL-6 indicating a strong association between DNA damage and inflammation. A time-course analysis showed that γH2AX and the several inflammatory cytokines mentioned above were activated simultaneously with all of them reaching maximum at ∼6 h. This was further confirmed by microarray analysis which revealed simultaneous up-regulation of both DNA damage and inflammatory pathways at 6 h. Concurrent treatment with cfCh inactivating agents, such as CNPs, DNase I and a novel DNA degrading agent namely resveratrol-copper (R-Cu), abrogated the activation of both H2AX and inflammatory cytokines further indicating that they are closely interrelated pathologies [9]. In an independent study, the same authors observed that when isolated cfDNA fragments were added to cultured cells, these could integrate into host cell genomes and activate both H2AX and NFκB [13]. Concurrent up-regulation of both these markers was again seen and to occur at 6 h. When the irradiated dying cancer cells were intravenously injected into mice, cfCh that were released from the dying cells could access nuclei of cells of target organs leading to their genomic integration [9]. Genomic integration of cfCh in vivo simultaneously activated both H2AX and multiple inflammatory cytokines in brain, liver, lung and heart of recipient animals [9]. Most significantly, γH2AX and NFκB fluorescent signals were found to co-localize in the same cellular nuclei further indicating that their activation was closely inter-linked (Fig. 2). NF-kB is normally sequestered in the cytoplasm in an inactived state bound to an inhibitor ƙB kinase complex [14]. Upon stressful stimuli, such as damage to DNA, NF-ƙB is activated leading to its translocation to the nucleus [15]. Although several NF-ƙB nuclear translocation sites have been described [16] finding in the above study of co-localization of fluorescent signals of γH2AX and NFκB prompts us to hypothesize that, upon DNA damage resulting from cfCh integration, the activated NFκB translocates specifically to the sites of cfCh integration and transactivates downstream genes in diverse locations. A schematic representation of this hypothesis is presented in Fig. 3. Administration of the cfCh inactivating agents namely CNPs, DNase

activated DDR proteins that include DNA-PKCs and DNA ligase-IV linkup the disperate cfCh fragments into long concatemers of discontinuous DNA segments which form novel substrates for integration into host cell genome by homologous and/or non-homologous recombination. Insertion of the dsDNA segments leads to deletions, rearrangements and instability of the recipient cell genome. A schematic representation of the above model is presented in Fig. 1 which also implicates activation of inflammation in the cells containing dsDNA breaks. A proposed mechanism by which inflammation might be activated is discussed below. Intravenous administration of cfDNA and cfCh isolated from human serum into mice led to their integration into nuclei of all organs and tissues examined as confirmed by FISH [8]. Genomic integration led to activation of H2AX and apoptotic responses in cells of multiple organs, and cfCh was again found to be more active than cfDNA. Fluorescent γH2AX signals could be visualized at the sites of their genomic integration as detected by immuno-FISH suggested that the act of their genomic integration was responsible for inflicting dsDNA breaks [8]. Concurrent treatment with DNase I and/or a cfCh neutralizing agent in the form of anti-histone antibody complexed nanoparticles (CNPs) abrogated both DNA damage and apoptotic responses. The authors proposed that circulating nucleic acids might represent a new class of intra-corporeal mobile genetic elements that act as continuously arising endogenous DNA mutagens [11,12]. The second study investigated the role of cfCh that are released from dying cells as fragmented chromosomal particles in inducing DNA damage in surrounding cells [9]. When irradiated human cancer cells were co-cultivated with mouse fibroblasts, numerous cfCh particles were found to emerge from the dying cells and to freely enter into the recipient cells to integrate into their genomes. This was followed by induction of both dsDNA breaks as evidenced by activation of H2AX and ATM. Interestingly, multiple inflammatory cytokines namely NFκB, IL-6, TNFα and IFNγ were also found to be up-regulated. More significantly, cells expressing γH2AX were found to co- express the 49


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Fig. 2. Co-localization of γH2AX and NFκB fluorescent signals in nuclei of vital organs of mice following intravenous injection of dying B16-F10 mouse melanoma cells (A). One hundred thousand cells were injected i.v. into mice and animals were sacrificed after 72 h. Control tissues do not show fluorescent signals (B). Reproduced from Cell Death Discovery 2017; 3: 17015.

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disease conditions including atherosclerosis, type 2 diabetes and Alzheimer’s disease as well as obesity and ageing [24]. That several cfCh inactivating agents were capable of preventing both dsDNA breaks and inflammation in vivo provides potential therapeutic options in chronic inflammatory conditions by preventing dsDNA breaks. Clearly, these initial results call for more intense investigations into the relationship between dsDNA breaks and inflammation which may have wide ranging implications for biology and medicine. Funding sources This study was supported by the Department of Atomic Energy, Government of India, through its grant CTCTMC to Tata Memorial Centre awarded to IM. Conflicts of interest None. Fig. 3. Schematic representation of proposed model to explain detection of co-localized fluorescent signals of γH2AX and NFκB.

References

I and R-Cu concurrently with dying cells in vivo, led to the abrogation of both γH2AX and NFκB indicating cfCh-induced dsDNA breaks was directly linked to the activation of inflammation [9]. Another clinically relevant study by the same group reported that toxicity of cancer chemotherapy is primarily caused by cfCh released from the initial round of drug-induced dying cells that trigger a cascading effect whereby the dying cells release more cfCh causing further rounds of dsDNA breaks and inflammation which exaggerate or amplify the toxic side effects [17]. Concurrent treatment with the cfCh inactivating agents mentioned above not only prevented dsDNA breaks but also prevented inflammation. It has been long known that presence of pathogens or foreign DNA in the cytoplasm can trigger immune activation [18,19]. DNA accumulation in the cytoplasm following genomic stress can trigger the DNA sensing cGAS-STING cytosolic pathway to trigger immune activation [20]. It has been recently reported that broken chromosomal fragments can mis-segregate during mitotic exit and lagging chromosomal fragments can recruit nuclear envelope components to form micronuclei. The latter can trigger genome instability-associated innate immune activation and that rupture of micronuclei envelope were essential in this process [21–23]. Results in the reference articles discussed above [8,9] suggest that micronuclei are not the only access route of DNA into the cytoplasm and presence of cytoplasmic micronuclei was not detected in any of the experiments. Rather, the authors of these articles detected the presence of DNA in the form of cfCh to be present not only in the cytoplasm but also in the nuclei of recipient cells [8,9]. Although the possibility cannot be excluded that intra-cellular cfCh with dsDNA breaks at each end may have activated DNA sensing cGAS-STING cytosolic pathway to trigger immune activation, the presence of cfCh in the nuclei and their co-localization with fluorescent γH2AX and NFκB foci suggests that dsDNA breaks may be the direct instigators of immune activation. dsDNA breaks and the resultant genetic stress could activate cytoplasmic NFκB to translocate to the cfCh integration sites marked by presence of phosphorylated H2AX, thereby explaining the detection of co-localized fluorescent signals (Fig. 3). Based on the above findings we propose that extranuclear cfCh, by their ability to inflict dsDNA breaks in healthy cells, act as continuously arising endogenous inflammogens and may have wide-ranging systemic effects. The afore-described studies also suggest that inflammatory cytokines represent a new family of DDR proteins that are activated following dsDNA breaks caused by genomic integration of cfCh. These findings are of particular relevance since DNA damage and inflammation are the underlying pathologies involved in multiple devastating

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