The many interactions between the innate immune system and the response to radiation

ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■

Contents lists available at ScienceDirect

Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Mini-review

The many interactions between the innate immune system and the response to radiation Serge M. Candéias a,b,c,*, Isabelle Testard a,b,c a b c

iRTSV-LCBM, CEA, Grenoble F-38000, France IRTSV-LCBM, CNRS, Grenoble F-38000, France iRTSV-LCBM, Univ. Grenoble Alpes, Grenoble F-38000, France

A R T I C L E

I N F O

Keywords: Innate immune system Inflammation Radiation response

A B S T R A C T

The role of the immune system in the protection of the organism against biological aggressions is long established and well-studied. A new role emerged more recently in the protection from – and the response to – physical trauma such as exposure to ionizing radiation. A pre-existing inflammation, induced by administration of an inflammatory cytokine or of a Toll-like receptor agonist, is indeed able to mitigate the toxic effects of acute radiation exposure. Conversely, it appears that the innate immune system can be activated during the course of the cellular response to radiation. Activation of different sensors and pattern recognition receptors by intra-cellular molecules such as HMGB1 or DNA released in the extracellular milieu or in the cytosol by irradiated cells induces the production of inflammatory and antiviral cytokines. In addition, in human monocytes and macrophages, the expression of inflammatory cytokine genes can be directly induced by p53- and ATM-dependent mechanisms. This last finding establishes a direct link between radiation-induced DNA damage response and radiation-induced inflammation. © 2015 Elsevier Ireland Ltd. All rights reserved.

Introduction: defense mechanisms at the cellular and tissue/organism levels Vertebrates have developed several types/layers of defense mechanisms to protect and maintain their integrity. At the cellular level, elaborate systems exist to preserve genetic material from injuries in order to preserve its functions and avoid the creation of mutations that could potentially result in cell transformation and endanger the whole organism [1]. For example, DNA double strand breaks (DSBs) are repaired by non-homologous end joining or homologous recombination depending on the position of the cell in the cell cycle. Oxidative base lesions are repaired by the base excision repair pathway, whereas bulky lesions distorting the DNA structure are repaired by the nucleotide excision repair pathway. The detection of DNA lesions by specific sensors activates an adequate cellular response that will culminate with the faithful restoration of the original genetic information. However, if the cells are unable to mount this response or face unrepairable DNA damage, they may choose to enter senescence or die by apoptosis to avoid the transmission of mutations. At the tissue/organism level, protection is enforced by the coordinated action of the various actors of the immune system [2]. Myeloid and lymphoid immune cells cooperate to provide an integrated array of defense mechanisms to

* Corresponding author. Tel. +33 4 38 78 92 49; fax: +33 4 37 78 54 87. E-mail address: [email protected] (S.M. Candéias).

maintain the tissue/organism integrity, structure and function. The role of the immune system and its components in the defense of the organism against infectious agents have being studied for decades. Myeloid cells such as neutrophils, dendritic cells, monocytes and macrophages constitute the so-called innate immune system and provide the first active line of defenses, while protection provided by B and T lymphocytes, essential components of the adaptive immune system, needs more time to be fully effective. The efficiency of the fight against pathogens relies on a tight cooperation between these two arms of the immune system. Tissueresident macrophages and dendritic cells sense the presence of pathogen-associated molecular patterns (PAMPs), i.e. microbial products and motives expressed by invading micro-organisms, via their pattern recognition receptors (PRRs), including the Toll-like receptors (TLRs). Activation of these receptors results in the production of inflammatory soluble mediators aimed at recruiting and activating on the site of infection more innate immune cells to contain the infection. If this innate immune response, which represents the first line of defense, is inefficient, then lymphocytes also recruited in situ will step in and mount an antigen specific response directed against the pathogen to facilitate its eradication. However, more recently, a new function emerged for the immune system, namely the protection of the organism against injuries resulting from physical trauma such as heat exposure [3], exposure to nanoparticular materials [4,5] or exposure to ionizing [6,7] or UV radiation [8]. This so-called sterile inflammation, by opposition to the classical infectious inflammation, is initiated by the detection

http://dx.doi.org/10.1016/j.canlet.2015.02.007 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Serge M. Candéias, Isabelle Testard, The many interactions between the innate immune system and the response to radiation, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.007

ARTICLE IN PRESS 2

S.M. Candéias, I. Testard/Cancer Letters ■■ (2015) ■■–■■

in the extracellular milieu of intracellular molecules [9–11]. These molecules, collectively referred to as danger signals, or Danger Associated Molecular Patterns (DAMPs) can also be recognized by, and activate PRRs. Their presence outside the cells denotes the occurrence of cellular damage with loss of cytoplasmic membrane integrity or death by a non-regulated mechanism able to activate the immune system [12–14]. The presence of inflammatory mediators released by injured cells most probably participates in the amplification of this phenomenon. Influence of the immune response on the response to radiation exposure In 1986, a report from Neta and collaborators showed for the first time that injection of a single dose of a recombinant cytokine has spectacular radioprotective effects [21]. Administration of recombinant IL-1 to mice subsequently exposed to 9.5 Gy of γ-radiation, a dose which results in the death of 100% of the mice in 17 days (LD100/17), was able to rescue animals from death. These experiments clearly showed that an inflammatory cytokine could protect mice from acute high dose radiation toxicity. In subsequent experiments, these authors showed that injection of IL-1α or TNFα one to 3 hours after irradiation with a LD95/30 dose could also induce dose dependent survival [22]. IL-1 was by far more efficient than TNFα in this assay using recombinant exogenous proteins. However, injection of neutralizing antibodies against IL-1 and TNFα receptors subsequently showed that endogenous levels of these cytokines were sufficient to confer significant radioprotection to exposed mice [23]. Thus, inflammatory mediators are able in vivo to activate signaling pathways leading to the induction of potent survival mechanisms able to rescue animals from radiation-induced death. Inflammatory cytokines are secreted following activation of PRRs by PAMPs and DAMPs, and in recent years, it was indeed found that TLR agonists can also provide radioprotection. For example, administration of LPS, a TLR4 ligand, protects against p53-dependent apoptosis of intestinal crypt cells [24], a mechanism partially dependent on TNFα/TNFR1 signaling [25]. TLR4 deficient mice exhibit an increased mortality rate 30 days after exposure to 5 Gy, a dose of radiation which does not affect the viability of TLR4-expressing animals [17]. More interestingly, injection of flagellin, a TLR5 ligand [18], or of a synthetic flagellin-derived polypeptide [19] was shown to protect mice from death following exposure to dose up to 13 Gy of ionizing radiation and even substantially increase survival of animals exposed to 17 Gy. This survival was flagellin dose-dependent, and required the expression of both TLR5 and MyD88, its intracellular signaling adapter [18,26]. The beneficial effects of flagellin were not due to a suppression of radiation-induced anemia and leukopenia, as administration did not prevent a strong decrease in leukocytes and red blood cells, and both B and T lymphocytes were found to be dispensable [18]. Flagellin-derived polypeptide was shown to be a potent suppressor of acute radiation toxicity effects such as apoptosis of gastrointestinal crypt cells and the subsequent loss of crypts, as well as loss of hematopoietic stem cells and early progenitors [19]. This last observation probably explains that the transfer of bone marrow cells from un-irradiated flagellintreated mice to irradiated mice is able to partially rescue radiationinduced mortality [18]. Similar results were obtained in a study addressing the role of TLR4 in the response to radiation, where the transfer of BM cells from un-irradiated mice injected with LPS 24 hours before harvesting increased the survival rate of 9 Gy irradiated recipient mice [17]. Injection of liposomes containing dichloromethylene bisphosphonate (Cl2-MBP) in mice results in a profound depletion of macrophages in their spleen and liver [16]. This treatment was found to accelerate radiation-induced death of mice exposed to 9.5 Gy, and the radioprotective effects of LPS administered before radiation-exposure were greatly attenuated (40%

Table 1 Summary of in-vivo experiments on the radioprotective effects of TRL ligands. TLR

TLR agonist

Organism

Radiation dosea

Ref

TLR2 TLR4 TLR4 TLR5 TLR5

Lipoprotein LPS LPS Flagellin Flagellin-derived peptide

9 Gy 9.5 Gy 5–13 Gy 8 Gy 9–13 Gy 6.5 Gy

[15] [16] [17] [18] [19]

TLR9

Synthetic immunomodulatory oligonucleotide

Mice Mice Mice Mice Mice Non-human primate Mice

8.4–10.4 Gy

[20]

a Radiation dose used for exposure and monitoring of survival at 30 days, except in non-human primate, for which survival at 40 days was monitored.

survival at 30 days in Cl2-MBP treated mice vs 80% in vehicle-only treated mice). The mitigation of radiation-induced gastrointestinal syndrome (reduction of crypt cells apoptosis, increase of crypt regeneration, recovery of intestinal absorption) induced by a TLR9 agonist was associated with an increased number of pericryptal macrophages [20]. Further, the survival of irradiated mice was increased when they were transplanted with macrophage cell line cells treated in vitro with this TLR9 agonist, supporting a role for macrophages in this phenomenon. However, using reciprocal BM chimera between wild type and TLR2-KO mice, the beneficial effects of a synthetic TLR2 lipopeptide ligand were found to be only partially dependent on TLR2 expression on BM-derived cells, whereas TLR2 expression on non-hematopoietic cells was required for the survival at 30 days of irradiated (9 Gy) mice [15]. Thus, hematopoietic cells are clearly not the only population involved in TLR-ligand induced prevention and/or mitigation of radiation toxicity. Indeed, the expression of TLRs is not restricted to BM-derived cell lineages and may be found on epithelial cells. Keratinocytes for example express several functional TLRs [27] and play in the skin the role of a sentinel able to detect all kinds of danger signals, microbial or non-infectious [28]. In any case, a role for TLR signaling in protection against radiation-induced cell death is clearly established. This protection requires TLR signaling through MyD88, even in experimental models where mice are only locally irradiated [29]. Of note, radioprotection can be provided by agonist of TLR2, TLR4, TLR5, TLR9 (Table 1), which use different signaling pathways [26,30]. Thus multiple inducers and more than one signaling pathway converge to achieve the same or similar effects. Do they operate through the same effectors? This point has not been directly addressed. Administration of TLR agonists induces a transient increase in several circulating cytokines, but, as no comprehensive monitoring was performed, it is not possible to extrapolate from the results of these studies. Indeed, the stimulation of PBLs by agonists of different TLRs clearly results in different patterns of pro-inflammatory gene expression [30]. In addition, TLRs show at least some cell type preference if not specificity for their expression among myeloid and non-myeloid lineages [26,31]. Each cell type will probably be able to produce a different array of pro-inflammatory cytokines following TLR activation, depending both on the collection of TLRs it expresses and on their signaling pathways [26,32]. It is therefore not possible today to precisely identify the active molecule(s) mediating these pro-survival effects. Several candidates have been proposed, such as IL-1, TNFα [21,23], IL-6 and G-CSF [33–35] to cite a few, but in fine, radioprotection results most likely from the coordinated action of several cytokines on multiple cell types. Nucleic acids as danger signals TLR signaling in myeloid cells has the ability to modulate radiation-induced responses through sterile inflammation and the production of inflammatory cytokines. Like the classical infectious

Please cite this article in press as: Serge M. Candéias, Isabelle Testard, The many interactions between the innate immune system and the response to radiation, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.007

ARTICLE IN PRESS S.M. Candéias, I. Testard/Cancer Letters ■■ (2015) ■■–■■

inflammatory reaction, this response is most likely aimed at stimulating a more efficient and coordinated response against radiation injury and the restoration of normal tissue homeostasis and function. It is induced by the detection of DAMPs released/produced from radiation-damaged cells, including nucleic acids, which can also be seen as danger signals. Multiple PRRs/sensors recognize and are activated by DNA outside the nucleus and evoke the production of inflammatory cytokines. This topic has been extensively reviewed recently [36–38] and only a brief overview will be provided here. TLR9 was the first described PRR specific for DNA. It is expressed in endosomes, predominantly in B lymphocytes and some dendritic cells, and has been originally described to be specific for unmethylated CpG motives found in bacterial DNA [39]. However, it later became evident that it can also be activated by mammalian DNA [40]. TLR9 signaling leads to the production of pro-inflammatory cytokines through the MyD88 pathway, but also to the production of type I interferons through activation of STING (stimulator of interferon genes), linking DNA recognition to the induction of an antiviral like response. Activation signals from several cytosolic DNA sensors (DAI, IFF116, MRE11, DNA–PK…) are integrated through STING [36–38], with the notable exceptions of AIM2 [41,42] and Rad50 [43], which cooperate to induce the production of proinflammatory IL-1β. On the one hand, Rad50 forms with dsDNA and Card9 a complex able to induce the NF-κB-dependent IL-1β gene expression [43]. On the other hand, upon sensing dsDNA, AIM2 participates in the formation of an inflammasome in which the procaspase 1 is activated and can therefore cleave the pro-IL-1β into the mature form of IL-1β to be secreted [41,42]. The common trigger for these DNA sensors is the abnormal accumulation of DNA in the cytosol, and the induced response is highly beneficial when this DNA originates from bacterial or viral infection. However, endogenous DNA also traffics to the cytosol, where it is constantly degraded by the cytoplasmic exonuclease TREX-1 [44]. Consequently, inactivation of TREX1 induces an accumulation of endogenous DNA fragments in the cytosol [45], and TREX1deficient mice develop autoimmune syndromes [46,47]. In humans, TREX mutations have been implicated in the development of Aicardi– Goutières syndrome, characterized by an increased level of circulating type I interferon [48]. Interestingly, the resolution of TREX1 crystal structure revealed that the active site of the enzyme cannot efficiently accommodate modified bases and DNA 3′ extremities [49]. This feature probably explains that oxidized DNA is less efficiently degraded by TREX1 and persists long enough to become a substrate for DNA sensor activation [50]. Of note, oxidized DNA can be imported within the cells. It is for example the case of DNA expelled by neutrophils in the form of the so-called neutrophil extracellular trap, which can be internalized and sensed as a danger signal by surrounding cells, probably as a way to amplify the inflammatory response [50]. It is therefore easy to extrapolate that oxidized/damaged DNA from cells dying from radiation exposure can also be captured in the same way if the dead/dying cells are not cleared properly, i.e. in a non-immunogenic way [12], linking cell death to innate immune response. Extracellular DNA, damaged or not, can also be detected and activate an innate immune response via its interaction with RAGE, the receptor for advanced glycation end products [51]. This cell surface receptor can directly recognize different forms of DNA in the extra-cellular milieu, free or complexed to the nucleosomal protein HMGB1, and deliver these cargos to the endosome, where nucleic acid sensing TLR3 and TLR9 are expressed [51]. HMGB1 is, in itself, a bona fide DAMP, and purified HMGB1 induces the secretion of pro-inflammatory cytokines in human monocytes and a strong inflammatory response in mice [52,53]. This protein interacts not only with RAGE but also with several TLRs, including TLR3 and TLR9. HMGB1 deficiency has been shown to impair their activation [54], but it is not clear yet if this modulation results from a synergy between RAGE and TLR3/TLR9

3

signaling or whether binding of nucleic acids to HMGB1 before delivery to endosomes provides a more potent stimulus. In any case, both capture and internalization of extracellular DNA and signaling through RAGE constitute additional ways to prompt the innate immune system to respond to the abnormal presence of DNA, whether it is oxidized or not. Like for DNA, sensing of intracellular RNA as a danger signal is achieved both in endosomes, via TLR3, and in the cytosol, through the activation of RIG-1 like receptors (RLRs) [55], and results in the production of type I interferons. Extracellular RNA is also recognized by RAGE [54]. Here again, HMGB1 seems to potentiate the response of TLR3 to its ligands [54], but the delivery of extracellular RNA to the endosome by RAGE was not investigated like it has been done for DNA [51]. RLRs have been described to recognize ssRNA and dsRNA of various sizes and in different structures [56], such as RNA molecules with a 5′ triphosphate end, or with U-rich tracts [56,57]. Interestingly, it was shown that UV exposure could generate ligands able to activate TLR3 from non-irradiated cells [58]. These ligands were identified as degradation products of the small nuclear U1 RNA molecules and consisted of short dsRNA. Hence, the uptake within the cell of self RNA motives can also initiate a sterile inflammatory response. This finding takes a special importance in light of the growing role attributed in intercellular communication to the transfer of mRNA and non-coding RNA, including miRNA, via micro-vesicles [59,60]. It is though that these mRNA and miRNA molecules can contribute to the response of the target cell by coding for new proteins or through post-transcriptional regulation of gene expression, respectively. They have been implicated not only in the modulation of immune responses [61], but also in the transmission of by-stander signals following radiationexposure [62,63]. However, to our knowledge, the effects of TLR3/ RLRs activation have never been thought in these experiments, and it is therefore not possible at that time to exclude that some of the effects attributed to exosomes result from exogenous RNA detection in host cells. Influence of the DNA damage response on immune activation Several proteins involved in the DNA damage response (DDR) elicited upon radiation exposure have been shown to also have a role in the sequence of events constituting a sterile inflammatory response. This is for example the case of Mre11 and Rad50. These molecules were identified as cytosolic DNA sensors [43,64], but are better known for their function as DSBs sensors in the initiation of the cascade of events leading to the activation of the DNA damage activated checkpoint. The MRN complex composed of Mre11, Rad50 and Nbs1 is indeed responsible for the initial detection of DSB in the nucleus. Its activation leads to the local recruitment and activation of ATM, which then phosphorylates its many substrates to enforce the DNA damage checkpoint and coordinate the cellular response to genotoxic stress [65,66]. These findings directly link the sensing of radiation-induced DNA damage in the nucleus and the initiation of an inflammatory response in the cytosol. Furthermore, they show that in both locations these proteins act in the very first stages of a complex reaction aimed at correcting an abnormal situation, i.e. the presence of damaged DNA and the presence of DNA outside the nucleus or the mitochondria, respectively. Similarly, the DNA–PK complex and its components, namely Ku70, Ku80 and the DNA–PKcs catalytic subunit [67], are also involved in DNA recognition both in the nucleus and the cytoplasm [68,69]. This complex is essential for DSB repair by non-homologous end-joining. The Ku heterodimer recognizes DNA stand breaks and recruits and activates DNA–PKcs [67]. The complex then protects DNA extremities from degradation and serves as a docking platform for the factors required for their processing and ligation [70,71]. The kinase activity

Please cite this article in press as: Serge M. Candéias, Isabelle Testard, The many interactions between the innate immune system and the response to radiation, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.007

ARTICLE IN PRESS 4

S.M. Candéias, I. Testard/Cancer Letters ■■ (2015) ■■–■■

of DNA–PKcs, activated after docking on the Ku heterodimer, is absolutely required for DNA end joining [72], as exemplified by the severe defect in DNA repair and the concomitant radiosensitivity observed in SCID mice, which harbor a kinase dead-DNA–PKcs mutant gene [73,74]. It then came as a surprise that the kinase activity of DNA–PK was not required for type I interferon induction following DNA sensing in the cytosol [68]. This finding may explain that Ku70 alone [69] or associated with Ku80 [68] can still trigger an immune reaction to cytosolic DNA, and demonstrate that the operating mechanisms are quite different in the cytosol and the nucleus. The detection and signalization of DSB through MRN or DNA– PK induce a coordinated cellular response which may include a cell cycle arrest, the induction of appropriate DNA repair mechanisms or the induction of cell death, depending on the cellular context and the extent of genotoxic stress. Implementation of the DNA damage dependent checkpoint requires the phosphorylation of numerous proteins which act as transducers of DNA damage detection or effectors to execute the cellular response. ATM is activated by several forms of genotoxic stress, including exposure to radiation, and plays a key role in the orchestration of this cascade of events [65,66]. Interestingly, several NF-κB family members such as IκB-α [75–78], p65 (RelA) [79] and NEMO [80] were identified among ATM substrates, suggesting that DNA damage-induced ATM activation can modulate NF-κB signaling. Indeed, IκB-α [77,78] or NEMO [80] phosphorylation allows the nuclear translocation of NF-κB and the activation of its DNA binding activity. Thus, even though it has not been directly tested, this activation cascade can potentially result in the induction of the transcription of NF-κB target genes, including pro-inflammatory cytokine genes, by a MyD88-like mechanism, and initiate a TLR-like inflammatory response. However, the recent discovery of p65 phosphorylation on Ser547 by ATM reveals another facet of the regulatory role of ATM on cytokine production. This last study indeed showed that p65 phosphorylation on Ser547 by ATM following genotoxic stress resulted in a weaker induction of the transcription of a group of NF-κB target genes involved in inflammation when compared to non-phosphorylable p65 protein where Ser547 was mutated to alanine [79]. This specific phosphorylation and its effects on gene expression have unfortunately only been tested after genotoxic stress, and not in the frame of classical inflammatory stimuli such as LPS or TNFα treatment. However, this finding raises the possibility that the panel of inflammatory mediators produced in different types of danger situations, and therefore the outcome of the inflammatory response, may be quantitatively and/ or qualitatively modulated by context dependent activation of NF-κB. The p53 tumor suppressor protein also plays a pivotal role in the DDR effector phase, mostly, but not exclusively, through its transcription factor activity. Upon genotoxic stress exposure, it becomes activated and induces the expression of numerous genes involved in the outcome of the response, including, but not limited to, cell cycle arrest, apoptosis, senescence. … P53 transactivation function varies according to the stimulus inducing its activation, the cell type in which it takes place and the genetic locus targeted [81,82]. However, beside these well-defined consequences of p53 activation, several types of genotoxic stress, including radiation exposure, induce a p53-dependent increase in TLR gene expression in human, but not murine, primary cells and cell lines [83]. Importantly, this transcriptional response does not require DSB per se, as it is observed in cells treated by Nutlin-3, an agonist of MDM2 that promotes accumulation of a transcriptionally active form of p53 by dissociating p53/MDM2 complexes [84]. This up-regulation of TLR gene expression renders the cells more responsive to stimulation by a TLR agonist. In a recent study, these authors now show that Nutlin-3 treatment induces a rapid and strong up-regulation of the expression of numerous pro-inflammatory genes, including cytokines and chemokines, in human primary monocytes and macrophages by a mechanism involving both p53 and NF-κB [85]. Thus, it appears

that in a situation of tissue damage, macrophages are able to integrate p53 and NF-κB signaling pathways to recruit neutrophils and induce a more potent inflammatory response to restore tissue homeostasis and function. Although this cooperation seems to take place only in monocytes and macrophages, it sheds new light on the intersection and interrelation of the signaling pathways elicited by genotoxic stress and immune stimuli. It must however be pointed out that the situation may be more complex. A comprehensive study of transcriptional changes following exposure of whole blood to low (0.05 Gy) and high (1 Gy) doses of radiation in vitro reported that low doses predominantly induced the regulation of genes involved in TLR, RLR, cytosolic DNA sensing, chemokine and cytokine signaling, whereas high dose exposure induced mainly genes involved in stress response and apoptosis [86]. A follow up study analyzing the response of isolated blood monocytes to the same doses reached essentially the same conclusions [87]. Low and high doses of radiation therefore induce rather more the NF-κB pathway or more p53-dependent signaling, respectively, in primary human leukocytes. Whether this dichotomy relies only on the amount and type of damage inflicted to the cells and/or on the level of ATM/p53 activation remains to be determined. Concluding remarks This review illustrates some of the many interconnections between the innate immune system and the response to radiation. We provided an overview of the effects of the activation of the innate immune system on the outcome of radiation exposure and how an innate immune response can be initiated by radiation exposure, either as a direct consequence of the activation of the DDR or by sensing danger signals such as nucleic acids released by damaged cells (Fig. 1). These interactions are probably modulated by several factors such as the level of activation of the DNA damage response, which probably shapes the response to low vs high doses of radiation, and the identity of the immune cells exposed to radiation as well as those sensing – and responding to – DAMPs. Indeed, the functional heterogeneity of the monocyte, macrophage

Ionizing radiation

DAMPs

TLRs / RAGE

HSPs HMGB1 TLR3 TLR9

p53 NF-kB Rad 50

Aim2 IFN STING IL-1β

Cytokines receptors

IL-1β Pro-inflammatory mediators

Cytokines

Nucleus

Fig. 1. Innate immune system activation following radiation-exposure. Irradiated cells (left) will be released in the extracellular milieu molecules usually located in their cytosol (heat shock proteins, HSPs), or in their nucleus (HMGB1, DNA). These molecules will act as danger signals and activate their cognate receptors (TLRs, RAGE) on neighboring cells. The downstream signaling pathways lead to the production of inflammatory cytokines. In the irradiated cells, DNA can also leak from the nucleus to the cytosol, where it can be sensed as a danger signal by various surveillance mechanisms. Several of them will lead to the production of type I interferons through STING. Rad50 and AIM2 will cooperate to induce the expression and maturation of IL-1β. Finally, in irradiated human monocytes and macrophages, p53 and NF-kB will cooperatively induce the expression of pro-inflammatory cytokines. All these secreted cytokines will activate their cognate receptors on neighboring cells. See text or details.

Please cite this article in press as: Serge M. Candéias, Isabelle Testard, The many interactions between the innate immune system and the response to radiation, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.007

ARTICLE IN PRESS S.M. Candéias, I. Testard/Cancer Letters ■■ (2015) ■■–■■

and dendritic cell subsets in the different tissues and differences in their expression of TLRs may result in the promotion of tissuespecific responses to DAMPs [31,88,89]. In addition, one must not forget that epithelial cells also express TLRs, adding to the diversity of responding cells. Importantly, the inflammatory response does not simply taper off but is actively curtailed by mechanisms involving both anti-inflammatory and pro-resolution mediators [90]. The timely expression of these mediators is crucial for the reversion to a normal physiological state, as exemplified by the now recognized importance of persistent inflammation in the development of many diseases, including cancer [91]. The mechanisms driving the resolution of radio-induced inflammation elicited by exposure to low and high doses of ionizing radiation remain to be identified. Conflict of interest None. References [1] S.P. Jackson, J. Bartek, The DNA-damage response in human biology and disease, Nature 461 (2009) 1071–1078. [2] R. Medzhitov, C.A. Janeway Jr., Decoding the patterns of self and nonself by the innate immune system, Science 296 (2002) 298–300. [3] H.M. Paterson, T.J. Murphy, E.J. Purcell, O. Shelley, S.J. Kriynovich, E. Lien, et al., Injury primes the innate immune system for enhanced Toll-like receptor reactivity, J. Immunol. 171 (2003) 1473–1483. [4] C. Dostert, V. Petrilli, R. Van Bruggen, C. Steele, B.T. Mossman, J. Tschopp, Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica, Science 320 (2008) 674–677. [5] V. Hornung, F. Bauernfeind, A. Halle, E.O. Samstad, H. Kono, K.L. Rock, et al., Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization, Nat. Immunol. 9 (2008) 847–856. [6] A. Cheda, E.M. Nowosielska, J. Wrembel-Wargocka, M.K. Janiak, Production of cytokines by peritoneal macrophages and splenocytes after exposures of mice to low doses of X-rays, Radiat. Environ. Biophys. 47 (2008) 275–283. [7] S.Z. Liu, S.Z. Jin, X.D. Liu, Y.M. Sun, Role of CD28/B7 costimulation and IL-12/IL-10 interaction in the radiation-induced immune changes, BMC Immunol. 2 (2001) 8. [8] S.M. Meeran, T. Punathil, S.K. Katiyar, IL-12 deficiency exacerbates inflammatory responses in UV-irradiated skin and skin tumors, J. Invest. Dermatol. 128 (2008) 2716–2727. [9] G.Y. Chen, G. Nunez, Sterile inflammation: sensing and reacting to damage, Nat. Rev. Immunol. 10 (2010) 826–837. [10] K.L. Rock, E. Latz, F. Ontiveros, H. Kono, The sterile inflammatory response, Annu. Rev. Immunol. 28 (2010) 321–342. [11] R.P. Wallin, A. Lundqvist, S.H. More, A. von Bonin, R. Kiessling, H.G. Ljunggren, Heat-shock proteins as activators of the innate immune system, Trends Immunol. 23 (2002) 130–135. [12] L. Galluzzi, J.M. Bravo-San Pedro, I. Vitale, S.A. Aaronson, J.M. Abrams, D. Adam, et al., Essential versus accessory aspects of cell death: recommendations of the NCCD 2015, Cell Death Differ. 22 (2014) 58–73. [13] H. Kono, K.L. Rock, How dying cells alert the immune system to danger, Nat. Rev. Immunol. 8 (2008) 279–289. [14] M. Li, D.F. Carpio, Y. Zheng, P. Bruzzo, V. Singh, F. Ouaaz, et al., An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells, J. Immunol. 166 (2001) 7128– 7135. [15] A.N. Shakhov, V.K. Singh, F. Bone, A. Cheney, Y. Kononov, P. Krasnov, et al., Prevention and mitigation of acute radiation syndrome in mice by synthetic lipopeptide agonists of Toll-like receptor 2 (TLR2), PLoS ONE 7 (2012) e33044. [16] C.A. Salkowski, R. Neta, T.A. Wynn, G. Strassmann, N. van Rooijen, S.N. Vogel, Effect of liposome-mediated macrophage depletion on LPS-induced cytokine gene expression and radioprotection, J. Immunol. 155 (1995) 3168–3179. [17] C. Liu, C. Zhang, R.E. Mitchel, J. Cui, J. Lin, Y. Yang, et al., A critical role of toll-like receptor 4 (TLR4) and its’ in vivo ligands in basal radio-resistance, Cell Death Dis. 4 (2013) e649. [18] M. Vijay-Kumar, J.D. Aitken, C.J. Sanders, A. Frias, V.M. Sloane, J. Xu, et al., Flagellin treatment protects against chemicals, bacteria, viruses, and radiation, J. Immunol. 180 (2008) 8280–8285. [19] L.G. Burdelya, V.I. Krivokrysenko, T.C. Tallant, E. Strom, A.S. Gleiberman, D. Gupta, et al., An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models, Science 320 (2008) 226–230. [20] S. Saha, P. Bhanja, L. Liu, A.A. Alfieri, D. Yu, E.R. Kandimalla, et al., TLR9 agonist protects mice from radiation-induced gastrointestinal syndrome, PLoS ONE 7 (2012) e29357. [21] R. Neta, S. Douches, J.J. Oppenheim, Interleukin 1 is a radioprotector, J. Immunol. 136 (1986) 2483–2485.

5

[22] R. Neta, J.J. Oppenheim, Cytokines in therapy of radiation injury, Blood 72 (1988) 1093–1095. [23] R. Neta, J.J. Oppenheim, R.D. Schreiber, R. Chizzonite, G.D. Ledney, T.J. MacVittie, Role of cytokines (interleukin 1, tumor necrosis factor, and transforming growth factor beta) in natural and lipopolysaccharide-enhanced radioresistance, J. Exp. Med. 173 (1991) 1177–1182. [24] K. Inagaki-Ohara, S. Yada, N. Takamura, M. Reaves, X. Yu, E. Liu, et al., p53dependent radiation-induced crypt intestinal epithelial cells apoptosis is mediated in part through TNF-TNFR1 system, Oncogene 20 (2001) 812–818. [25] T.E. Riehl, R.D. Newberry, R.G. Lorenz, W.F. Stenson, TNFR1 mediates the radioprotective effects of lipopolysaccharide in the mouse intestine, Am. J. Physiol. Gastrointest. Liver Physiol. 286 (2004) G166–G173. [26] T. Kawai, S. Akira, The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors, Nat. Immunol. 11 (2010) 373–384. [27] M.C. Lebre, A.M. van der Aar, L. van Baarsen, T.M. van Capel, J.H. Schuitemaker, M.L. Kapsenberg, et al., Human keratinocytes express functional Toll-like receptor 3, 4, 5, and 9, J. Invest. Dermatol. 127 (2007) 331–341. [28] F.O. Nestle, P. Di Meglio, J.Z. Qin, B.J. Nickoloff, Skin immune sentinels in health and disease, Nat. Rev. Immunol. 9 (2009) 679–691. [29] W.J. Brickey, I.P. Neuringer, W. Walton, X. Hua, E.Y. Wang, S. Jha, et al., MyD88 provides a protective role in long-term radiation-induced lung injury, Int. J. Radiat. Biol. 88 (2012) 335–347. [30] E. Eldering, C.A. Spek, H.L. Aberson, A. Grummels, I.A. Derks, A.F. de Vos, et al., Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways, Nucleic Acids Res. 31 (2003) e153. [31] M. Merad, P. Sathe, J. Helft, J. Miller, A. Mortha, The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting, Annu. Rev. Immunol. 31 (2013) 563–604. [32] R. Barbalat, L. Lau, R.M. Locksley, G.M. Barton, Toll-like receptor 2 on inflammatory monocytes induces type I interferon in response to viral but not bacterial ligands, Nat. Immunol. 10 (2009) 1200–1207. [33] V.I. Krivokrysenko, A.N. Shakhov, V.K. Singh, F. Bone, Y. Kononov, I. Shyshynova, et al., Identification of granulocyte colony-stimulating factor and interleukin-6 as candidate biomarkers of CBLB502 efficacy as a medical radiation countermeasure, J. Pharmacol. Exp. Ther. 343 (2012) 497–508. [34] R. Neta, J.J. Oppenheim, S.D. Douches, Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocytemacrophage colony-stimulating factor, J. Immunol. 140 (1988) 108–111. [35] R. Neta, R. Perlstein, S.N. Vogel, G.D. Ledney, J. Abrams, Role of interleukin 6 (IL-6) in protection from lethal irradiation and in endocrine responses to IL-1 and tumor necrosis factor, J. Exp. Med. 175 (1992) 689–694. [36] G. Chatzinikolaou, I. Karakasilioti, G.A. Garinis, DNA damage and innate immunity: links and trade-offs, Trends Immunol. 35 (2014) 429–435. [37] C.K. Holm, S.R. Paludan, K.A. Fitzgerald, DNA recognition in immunity and disease, Curr. Opin. Immunol. 25 (2013) 13–18. [38] S.R. Paludan, A.G. Bowie, Immune sensing of DNA, Immunity 38 (2013) 870–880. [39] H. Hemmi, O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, et al., A toll-like receptor recognizes bacterial DNA, Nature 408 (2000) 740–745. [40] F.J. Barrat, T. Meeker, J. Gregorio, J.H. Chan, S. Uematsu, S. Akira, et al., Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus, J. Exp. Med. 202 (2005) 1131–1139. [41] T. Fernandes-Alnemri, J.W. Yu, P. Datta, J. Wu, E.S. Alnemri, AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA, Nature 458 (2009) 509–513. [42] V.A. Rathinam, Z. Jiang, S.N. Waggoner, S. Sharma, L.E. Cole, L. Waggoner, et al., The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses, Nat. Immunol. 11 (2010) 395–402. [43] S. Roth, A. Rottach, A.S. Lotz-Havla, V. Laux, A. Muschaweckh, S.W. Gersting, et al., Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1beta production, Nat. Immunol. 15 (2014) 538–545. [44] T. Lindahl, D.E. Barnes, Y.G. Yang, P. Robins, Biochemical properties of mammalian TREX1 and its association with DNA replication and inherited inflammatory disease, Biochem. Soc. Trans. 37 (2009) 535–538. [45] J. Ahn, P. Ruiz, G.N. Barber, Intrinsic self-DNA triggers inflammatory disease dependent on STING, J. Immunol. 193 (2014) 4634–4642. [46] M. Morita, G. Stamp, P. Robins, A. Dulic, I. Rosewell, G. Hrivnak, et al., Genetargeted mice lacking the Trex1 (DNase III) 3′→5′ DNA exonuclease develop inflammatory myocarditis, Mol. Cell. Biol. 24 (2004) 6719–6727. [47] D.B. Stetson, J.S. Ko, T. Heidmann, R. Medzhitov, Trex1 prevents cell-intrinsic initiation of autoimmunity, Cell 134 (2008) 587–598. [48] P. Lebon, J. Badoual, G. Ponsot, F. Goutieres, F. Hemeury-Cukier, J. Aicardi, Intrathecal synthesis of interferon-alpha in infants with progressive familial encephalopathy, J. Neurol. Sci. 84 (1988) 201–208. [49] U. de Silva, S. Choudhury, S.L. Bailey, S. Harvey, F.W. Perrino, T. Hollis, The crystal structure of TREX1 explains the 3′ nucleotide specificity and reveals a polyproline II helix for protein partnering, J. Biol. Chem. 282 (2007) 10537– 10543. [50] N. Gehrke, C. Mertens, T. Zillinger, J. Wenzel, T. Bald, S. Zahn, et al., Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing, Immunity 39 (2013) 482–495. [51] C.M. Sirois, T. Jin, A.L. Miller, D. Bertheloot, H. Nakamura, G.L. Horvath, et al., RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA, J. Exp. Med. 210 (2013) 2447–2463.

Please cite this article in press as: Serge M. Candéias, Isabelle Testard, The many interactions between the innate immune system and the response to radiation, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.007

ARTICLE IN PRESS 6

S.M. Candéias, I. Testard/Cancer Letters ■■ (2015) ■■–■■

[52] U. Andersson, H. Wang, K. Palmblad, A.C. Aveberger, O. Bloom, H. Erlandsson-Harris, et al., High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes, J. Exp. Med. 192 (2000) 565–570. [53] G.P. Sims, D.C. Rowe, S.T. Rietdijk, R. Herbst, A.J. Coyle, HMGB1 and RAGE in inflammation and cancer, Annu. Rev. Immunol. 28 (2010) 367–388. [54] H. Yanai, T. Ban, Z. Wang, M.K. Choi, T. Kawamura, H. Negishi, et al., HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses, Nature 462 (2009) 99–103. [55] Y.M. Loo, M. Gale Jr., Immune signaling by RIG-I-like receptors, Immunity 34 (2011) 680–692. [56] M. Schlee, Master sensors of pathogenic RNA – RIG-I like receptors, Immunobiology 218 (2013) 1322–1335. [57] C.A. Hagmann, A.M. Herzner, Z. Abdullah, T. Zillinger, C. Jakobs, C. Schuberth, et al., RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells, PLoS ONE 8 (2013) e62872. [58] J.J. Bernard, C. Cowing-Zitron, T. Nakatsuji, B. Muehleisen, J. Muto, A.W. Borkowski, et al., Ultraviolet radiation damages self noncoding RNA and is detected by TLR3, Nat. Med. 18 (2012) 1286–1290. [59] G. Cheng, Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy, Adv. Drug Deliv. Rev. 81C (2014) 75–93. [60] J.G. van den Boorn, J. Dassler, C. Coch, M. Schlee, G. Hartmann, Exosomes as nucleic acid nanocarriers, Adv. Drug Deliv. Rev. 65 (2013) 331–335. [61] P.D. Robbins, A.E. Morelli, Regulation of immune responses by extracellular vesicles, Nat. Rev. Immunol. 14 (2014) 195–208. [62] A.H. Al-Mayah, S.L. Irons, R.C. Pink, D.R. Carter, M.A. Kadhim, Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation, Radiat. Res. 177 (2012) 539–545. [63] K.K. Jella, S. Rani, L. O’Driscoll, B. McClean, H.J. Byrne, F.M. Lyng, Exosomes are involved in mediating radiation induced bystander signaling in human keratinocyte cells, Radiat. Res. 181 (2014) 138–145. [64] T. Kondo, J. Kobayashi, T. Saitoh, K. Maruyama, K.J. Ishii, G.N. Barber, et al., DNA damage sensor MRE11 recognizes cytosolic double-stranded DNA and induces type I interferon by regulating STING trafficking, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 2969–2974. [65] M.F. Lavin, S. Kozlov, ATM activation and DNA damage response, Cell Cycle 6 (2007) 931–942. [66] T.H. Stracker, I. Roig, P.A. Knobel, M. Marjanovic, The ATM signaling network in development and disease, Front. Genet. 4 (2013) 37. [67] T.M. Gottlieb, S.P. Jackson, The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen, Cell 72 (1993) 131–142. [68] B.J. Ferguson, D.S. Mansur, N.E. Peters, H. Ren, G.L. Smith, DNA-PK is a DNA sensor for IRF-3-dependent innate immunity, eLife 1 (2012) e00047. [69] X. Zhang, T.W. Brann, M. Zhou, J. Yang, R.M. Oguariri, K.B. Lidie, et al., Cutting edge: Ku70 is a novel cytosolic DNA sensor that induces type III rather than type I IFN, J. Immunol. 186 (2011) 4541–4545. [70] S. Costantini, L. Woodbine, L. Andreoli, P.A. Jeggo, A. Vindigni, Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK, DNA Repair (Amst) 6 (2007) 712–722. [71] E. Weterings, D.J. Chen, DNA-dependent protein kinase in nonhomologous end joining: a lock with multiple keys?, J. Cell Biol. 179 (2007) 183–186. [72] W.D. Block, Y. Yu, D. Merkle, J.L. Gifford, Q. Ding, K. Meek, et al., Autophosphorylation-dependent remodeling of the DNA-dependent protein

[73]

[74] [75] [76]

[77]

[78]

[79]

[80]

[81] [82] [83]

[84]

[85]

[86]

[87]

[88] [89] [90]

[91]

kinase catalytic subunit regulates ligation of DNA ends, Nucleic Acids Res. 32 (2004) 4351–4357. T. Blunt, N.J. Finnie, G.E. Taccioli, G.C. Smith, J. Demengeot, T.M. Gottlieb, et al., Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation, Cell 80 (1995) 813–823. G.C. Bosma, R.P. Custer, M.J. Bosma, A severe combined immunodeficiency mutation in the mouse, Nature 301 (1983) 527–530. M. Jung, A. Kondratyev, S.A. Lee, A. Dimtchev, A. Dritschilo, ATM gene product phosphorylates I kappa B-alpha, Cancer Res. 57 (1997) 24–27. M. Jung, S.A. Lee, Y. Zhang, A. Dritschilo, Regulation of p53 in response to ionizing radiation in ataxia telangiectasia fibroblasts, Int. J. Radiat. Oncol. Biol. Phys. 37 (1997) 417–422. S.J. Lee, A. Dimtchev, M.F. Lavin, A. Dritschilo, M. Jung, A novel ionizing radiation-induced signaling pathway that activates the transcription factor NF-kappaB, Oncogene 17 (1998) 1821–1826. B. Piret, S. Schoonbroodt, J. Piette, The ATM protein is required for sustained activation of NF-kappaB following DNA damage, Oncogene 18 (1999) 2261– 2271. H. Sabatel, E. Di Valentin, G. Gloire, F. Dequiedt, J. Piette, Y. Habraken, Phosphorylation of p65(RelA) on Ser(547) by ATM represses NF-kappaBdependent transcription of specific genes after genotoxic stress, PLoS ONE 7 (2012) e38246. Z.H. Wu, Y. Shi, R.S. Tibbetts, S. Miyamoto, Molecular linkage between the kinase ATM and NF-kappaB signaling in response to genotoxic stimuli, Science 311 (2006) 1141–1146. R. Beckerman, C. Prives, Transcriptional regulation by p53, Cold Spring Harb. Perspect. Biol. 2 (2010) a000935. S. Sengupta, C.C. Harris, p53: traffic cop at the crossroads of DNA repair and recombination, Nat. Rev. Mol. Cell Biol. 6 (2005) 44–55. D. Menendez, M. Shatz, K. Azzam, S. Garantziotis, M.B. Fessler, M.A. Resnick, The Toll-like receptor gene family is integrated into human DNA damage and p53 networks, PLoS Genet. 7 (2011) e1001360. L.T. Vassilev, B.T. Vu, B. Graves, D. Carvajal, F. Podlaski, Z. Filipovic, et al., In vivo activation of the p53 pathway by small-molecule antagonists of MDM2, Science 303 (2004) 844–848. J.M. Lowe, D. Menendez, P.R. Bushel, M. Shatz, E.L. Kirk, M.A. Troester, et al., p53 and NF-kappaB coregulate proinflammatory gene responses in human macrophages, Cancer Res. 74 (2014) 2182–2192. H. El-Saghire, H. Thierens, P. Monsieurs, A. Michaux, C. Vandevoorde, S. Baatout, Gene set enrichment analysis highlights different gene expression profiles in whole blood samples X-irradiated with low and high doses, Int. J. Radiat. Biol. 89 (2013) 628–638. H. El-Saghire, A. Michaux, H. Thierens, S. Baatout, Low doses of ionizing radiation induce immune-stimulatory responses in isolated human primary monocytes, Int. J. Mol. Med. 32 (2013) 1407–1414. S. Gordon, P.R. Taylor, Monocyte and macrophage heterogeneity, Nat. Rev. Immunol. 5 (2005) 953–964. P.J. Murray, T.A. Wynn, Protective and pathogenic functions of macrophage subsets, Nat. Rev. Immunol. 11 (2011) 723–737. C.N. Serhan, S.D. Brain, C.D. Buckley, D.W. Gilroy, C. Haslett, L.A. O’Neill, et al., Resolution of inflammation: state of the art, definitions and terms, FASEB J. 21 (2007) 325–332. C. Nathan, A. Ding, Nonresolving inflammation, Cell 140 (2010) 871–882.

Please cite this article in press as: Serge M. Candéias, Isabelle Testard, The many interactions between the innate immune system and the response to radiation, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.007