‘From R-lupus to cancer’: Reviewing the role of R-loops in innate immune responses

Cells possess an inherent and evolutionarily conserved ability to detect and respond to the presence of foreign and pathological ‘self ’ nucleic acids. The result is the stimulation of innate immune responses, signalling to the host immune system that defence mechanisms are necessary to protect the organism. To date, there is a vast body of literature describing innate immune responses to various nucleic acid species, including dsDNA, ssDNA and ssRNA etc., however, there is limited information available on responses to R-loops. R-loops are 3-stranded nucleic acid structures that form during transcription, upon DNA damage and in various other settings. Emerging evidence suggests that innate immune responses may also exist for the detection of R-loop related nucleic acid structures, implicating R-loops as drivers of inflammatory states. In this review, we aim to summarise the evidence indicating that R-loops are immunogenic species that can trigger innate immune responses in physiological and pathological settings and discuss the implications of this in the study of various diseases and therapeutic development.


Introduction
Nucleic acid (NA) immunity is becoming an increasingly popular field of study.NAs are highly immunostimulatory and the ability of an organism to distinguish between self and foreign NAs is critical in host defence, with the presence of non-self-molecules leading to protective measures including the stimulation of an immune response.However, it is arguably equally as important for an organism to detect and respond to pathogenic self NAs, as these are indicators of failing cellular integrity [1,2].Indeed, it is well documented that mammalian cells can sense the presence of pathogenic NA structures and mount appropriate defensive responses.When an organism's ability to detect NAs goes awry, it can lead to devastating consequences, including the development of autoimmune diseases such as Aicardi-Goutières syndrome and systemic lupus erythematosus [1,3,4].R-loops are quaternary nucleic acid structures that arise naturally within cells as inherent by-products of transcription when nascent RNA binds to template DNA (Fig. 1A).This leads to the creation of a transient 3 stranded structure, consisting of a DNA-RNA hybrid and a displaced single strand of DNA behind RNA polymerases [5].R-loops are normal features that have been found to perform many physiological roles within cells including transcription regulation and DNA repair and are generally referred to as 'scheduled' R-loops [6].R-loops can also form at heterochromatic regions such as centromeres and telomeres and can be induced by DNA damage [5], the latter of which is a type of 'unscheduled' R-loop, which can have detrimental consequences, such as genomic instability [7].
To avoid unscheduled R-loops, cells employ sophisticated mechanisms to regulate their formation, maintenance, and resolution (reviewed extensively elsewhere [5][6][7]).When this fails, unscheduled R-loops arise that have been found to play pathological roles and may lead to genomic instability through deregulation of DNA replication, transcription, and repair.Recently, de-regulated R-loop processing has been linked to the stimulation of immune responses, with studies showing that they can be the source of immunogenic nucleic acid by-products including ssDNA and DNA-RNA hybrids (Fig 1B-C).This raises exciting prospects for the exploitation of these cellular structures in understanding disease pathology and in therapeutic development.

Innate sensing of nucleic acids
The ability of a cell to distinguish between self and non-self NAs is an ☆ This Special Issue is edited by Jessica Downs.or from natural aging, leads to the accumulation of R-loops and subsequently, ssDNA fragments, which are actively transported into the cytoplasm in an RPA-dependent manner.Cytoplasmic ssDNA is sensed by an unconfirmed sensor (*thought to be cGAS based on STING activation) and leads to the nuclear translocation of NF-kB and the activation of downstream inflammatory signalling [9].(Right) Aicardi-Goutières Syndrome (AGS) is a neuroinflammatory disease with features mimicking congenital viral infection.Mutations in RNASEH2 are detectable in > 50% of cases and have been found to cause the build-up of co-transcriptional R-loops which are processed by XPG and XPF, as well as an increase in micronuclei formation.Whilst it is yet to be confirmed if R-loop specific by-products are contained in these micronuclei, or if other intermediary species exist (such as DNA-RNA hybrids) following processing, R-loop accumulation has been linked to activation of NLRP3 inflammasome and cGAS/STING signalling [10,11].(C) The detection and response to pathogenic R-loop by-products leads to the activation of downstream inflammatory signalling mediated by various transcription factors such as IRF3 and NF-kB.Created with BioRender.com.evolutionarily conserved trait that is critical in host defence from infection and responses to pathogenic self NAs have been implicated in diseases such as cancer, autoimmunity and neurodegeneration [12][13][14][15].Several highly sophisticated sensors, including pattern recognition receptors (PRRs), and their subsequent pathways have been identified, with roles in NA sensing and response.These pathways have been reviewed extensively elsewhere [1,12,13,16,17], but here we provide a brief overview of those sensors and their downstream signalling pathways that have been particularly implicated in the detection of pathogenic self NAs, and have emerging roles in R-loop detection.

Toll-like receptors (TLRs)
Toll-Like Receptors (TLRs) are the most well characterised mammalian PRRs to date.Several TLRs can sense a plethora of pathogenic features common in microorganisms and are thus critical inducers of innate and adaptive immunity in response to infection.However, a subset of TLRs exist that are activated by various nucleic acid structures, including TLR3, TLR7, TLR8, TLR9 and TLR13 [18,19].These innate sensors are located at the cell membrane or in endosomes.Whilst some have been linked to responses to dsRNA (TLR3) and ssRNA (TLR7/8/13), TLR9 is the most well characterised innate DNA sensor [19].TLR9 is a sensor for pathogenic ssDNA, it is located at endosomes and predominantly expressed in human phagocytes and B cells [20].Interestingly, studies which tethered TLR9 to the plasma membrane demonstrated its ability to respond to self-DNA, highlighting the importance of subcellular localisation in the regulation of NA responses [21].Activation of TLRs by self-NAs leads to downstream activation of inflammatory signalling including Type I IFN, NFkB and TNFα responses, as well as others [1].

cGAS/STING signalling
The cGAMP synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway is widely regarded as the principal mammalian mechanism of dsDNA sensing [22,23].Activation of this pathway occurs when cGAS binds to intracellular foreign or host-derived dsDNA, localised within the cytoplasm or in micronuclei, leading to the production of the second messenger molecule, 2′3′cyclicGMP-AMP (cGAMP).cGAMP is a potent activator of STING, which it binds to and activates at the endoplasmic reticulum membrane.Activated STING is then trafficked to the Golgi apparatus, where it recruits and activates TANK-binding kinase 1 (TBK1), leading to further recruitment of interferon regulatory factor 3 (IRF3).IRF3, in turn, translocates to the nucleus and stimulates the expression of its downstream mediators which include several interferon stimulated genes (ISGs) and chemokines [24].Activated STING can also induce downstream NFkB signalling [25], leading to further inflammatory signalling.

Inflammasomes
Nucleic acids can also be sensed and responded to by macromolecular signalling complexes known as inflammasomes.Inflammasomes assemble in the cytoplasm in response to infection or stress stimuli and they typically contain a sensor, an adapter, and an effector [26].Several inflammasomes have been identified to date, but the best characterised NA sensing inflammasomes include the NLRP3 (nucleotide-binding oligomerization-like receptor family, pyrin domain-containing protein 3), AIM2 (absent in myeloma 2) and IFI16 (IFNγ-inducible protein 16) inflammasomes [27].Activation of inflammasomes leads to downstream activation of pro-inflammatory cytokines (including 1 L-1β and IL-18) and, usually, subsequent cell death [26].

DExD/H box helicases
There is a growing appreciation for the role of helicases in the mediation of immune responses to NAs.DExD/H box proteins are a conserved, large superfamily of RNA helicases that share eight conserved motifs and are involved in all aspects of RNA regulation and metabolism including transcription, splicing, translation and more [28,29].Several DExD/H-box helicases have been implicated in anti-viral immunity as sensors of viral nucleic acids, including RIG-I, DDX41 and MDA5 amongst numerous others [28], and are considered bona-fide PRRs.Other studies have found that these helicases can function downstream to PRRs, regulating anti-viral immune signalling at various levels [28].
The evolution of multiple, highly sophisticated mechanisms to sense and respond to NAs clearly indicates the importance of this ability.From herein we will discuss those sensors that have been implicated in R-loop detection and the resulting downstream consequences.

R-loops, their regulators, and innate immune responses
Several reports have emerged implicating R-loops and their byproducts in innate immune activation.Early studies demonstrated the ability of synthetic DNA-RNA hybrids to directly bind to and activate cGAS [30], whilst TLR9 and NLRP3 were also shown to be capable of potentiating responses to viral and bacterial DNA-RNA hybrids, respectively [31,32].Since then, several reports have surfaced implicating R-loops/DNA-RNA hybrids as immunogenic NA species.

R-loops and their by-products as mediators of inflammation
The ability of R-loops/DNA-RNA hybrids to directly induce inflammatory responses in healthy, physiological settings has recently been demonstrated.R-loop dynamics have been linked to the differentiation of human pluripotent stem cells and recent studies have implicated their homeostasis in the development of haematopoietic stem and progenitor cells (HSPCs) [33,34].HSPC numbers must be tightly regulated to ensure sufficient haematopoiesis whilst avoiding excessive expansion, which is associated with haematologic malignancies.Inflammatory signalling has been shown to play a role in maintaining this balance [35,36].Weinreb et al., recently uncovered that DDX41 (DEAD-box helicase 41) is an important orchestrator of HSPC homeostasis [34], with loss of DDX41 expression leading to R-loop accumulation and an associated induction of cGAS/STING mediated inflammatory signalling.Crucially, this leads to an increase in HSPC production.These data implicate an R-loop driven inflammatory cascade in physiological settings.
Evidence is accumulating that there is a fine balance between this and the onset of diseased states, as is seen in when deregulated haematopoiesis leads to the formation of myelodysplastic syndromes (MDS) which are known to display aberrant immune activation in HSPCs, including constitutive activation of the NLRP3 inflammasome [37].Mechanistically, McLemore et al., demonstrated that unresolved R-loops accumulate both in the nucleus and cytosol of MDS model cell lines and that the cytosolic population can illicit immune responses via engagement of cGAS.Subsequent signalling through STING/NLRP3 was confirmed in these models [38].Furthermore, they demonstrated that these R-loops are predominantly the result of elevated RNA polymerase III (RNAP3) activity in MDS samples and that inhibition of RNAP3 can cause significant downregulation of R-loops and, in turn, select ISGs.
Immunogenic cytosolic R-loop species are becoming increasingly evident in the literature.Through a set of elegant experiments, Crossley et al., demonstrated that by-products of R-loop processing are responsible for inducing an innate immune response in cells (Fig. 1B & 1 C) [8].The authors showed that loss of the R-loop resolving proteins, SETX (senataxin) or BRCA1, led to the accumulation of nuclear R-loops that are processed in an XPG/F (xeroderma pigmentosum group G/F-complementing protein) dependent manner to form DNA-RNA hybrids.These hybrids are actively exported to the cytoplasm where they accumulate and trigger downstream IRF3 signalling via direct sensing by the PRRs cGAS and TLR3.Notably, the authors also sequenced the R-loop species and whilst they found that the DNA-RNA hybrids arose from genomic R-loops, only a small subset of sequences were identified in the cytoplasmic fraction, indicating that the elevated R-loops were not uniformly processed and that some may be more susceptible to processing than others.This study also definitively linked pathologically driven R-loops to IRF3-linked immune signalling.BRCA1 mutant ovarian cancer cells and SETX mutant AOA2 (ataxia oculomotor apraxia type 2, a neurodegenerative disease) cells both displayed accumulation of R-loop derived cytoplasmic DNA-RNA hybrids which were linked with stimulation of ISGs, whilst knockdown of SAMHD1 (which is responsible for the autoimmune disease AGS) displayed similar phenotypes.
DNA damage and related inflammatory signalling is well documented as a driver of aging [39].Recent reports have also implicated R-loop by-products in the process of 'inflammaging', with accumulating R-loops being observed as aging progresses (Fig. 1B) [40,41].ERCC1 (excision repair cross complementation group 1) and XPF form a heterodimeric endonuclease that is involved in the nucleotide excision repair (NER) pathway [42] and mutations in ERCC1 and XPF cause symptoms of the premature aging-like progeroid syndrome, XFE [43,44].Interestingly, recent studies on a mouse model of XFE showed that chronic DNA damage underlies aging related degeneration of the pancreas in an R-loop dependent manner.Ercc1 -/-animals displayed heightened levels of R-loops, attributed to an inability to repair DNA lesions, but likely compounded by a loss of ERCC1mediated resolution of R-loops.Elevated R-loop levels led to the release and cytoplasmic build-up of immunogenic ssDNA fragments, potentially being sensed through cGAS/STING signalling [9].Chatzidoukaki et al., also noted heightened levels of cytoplasmic DNA-RNA hybrids in their experiments, and although likely immunogenic R-loop derived species, they did not expand upon their source or effects (Fig. 1B & 1 C).Furthermore, this study demonstrated a sophisticated, rationalised therapeutic strategy targeting these DNA-RNA hybrids, which led to the reversal of chronic pancreatitis symptoms and highlighting the vast potential in targeting these inflammatory species.

R-loops as immunogenic species in disease
Some of the most compelling evidence supporting a role for R-loops as drivers of inflammatory responses comes from the study of autoimmune diseases.Aicardi-Goutières Syndrome (AGS) is a rare, inherited autoimmune disease which leads to progressive neurological damage driven by inflammation [45].Studies have shown that AGS patients display aberrant accumulation of DNA-RNA hybrids in intergenic regions [11,46], implicating these NA species in inappropriate activation of immune responses.AGS has been attributed to mutations in several genes including RNASEH2, TREX1 and SAMHD1, all of which are specialised nucleases implicated in R-loop processing [45,47].RNASEH2 is an endonuclease that can degrade RNA within DNA-RNA hybrids and has been shown to be directly involved in the resolution of co-transcriptional R-loops [10].AGS-related mutations in RNASEH2 have been linked to induction of cGAS/STING signalling [48] and, consequently, loss of RNASEH2 has been directly implicated in the induction of inflammatory signalling in AGS in a processed R-loop dependent manner (Fig. 1B & 1 C) [10].Furthermore, AGS model cell lines harbouring TREX1 or RNASEH2 mutations have been shown to induce R-loop and micronuclei linked inflammatory DNA damage, capable of inducing downstream STING and NLRP3 signalling [11,49].Interestingly, loss of RNASEH2 in studies of AGS demonstrated an accumulation of R-loops only at specific sequence sites, namely short, intronless genes [10], illustrating the complex nature of R-loop regulation and raising the possibility that different R-loop species can induce variable downstream outputs.
Mutations in RNASEH2 and TREX1 have also been reported in the autoimmune diseases, systemic lupus erythematosus (SLE) and Familial Chilblain Lupus (FCL) [50][51][52], whilst SAMHD1 mutations have been shown to induce an enrichment of R-loops [53].However, definitive evidence is lacking to link these diseases to R-loops and SAMHD1-loss driven R-loops to inflammation, but given the available evidence, it seems highly likely that these links will be made.Together, these findings strongly indicate that disrupted R-loop homeostasis is a driving factor of pathology in various autoimmune disease and more work is required to definitively characterise the underlying mechanisms.
Evidence that R-loop species also contribute to inflammatory skin conditions is emerging, with studies into the chronic inflammatory skin disease, psoriasis, uncovering high levels of cytoplasmic NA species in diseased keratinocytes.Subsequent studies have demonstrated that these NAs include DNA-RNA hybrids that are enriched in psoriatic lesions, potentially from heightened levels of reverse transcriptase activity [54].Ultraviolet (UV) light exposure can cause skin injury and lead to the release of inflammatory cytokines [55].Transcriptomic analysis of UV-damaged versus healthy skin revealed that UV-induced inflammatory cascades are mediated by the formation of the 'HSF4-COIL' transcription factor complex which leads to the accumulation of immunogenic R-loops [56], further implicating these NA species in inflammatory skin conditions.
Taken together, these findings from a range of human disease states provides compelling evidence that R-loops are immunogenic species that can drive inflammation and disease aetiology, including cancer, which will be discussed in detail later.

R-loop regulators & links to inflammation
Whilst compelling data is accumulating on the direct impact of Rloops and their by-products on inflammatory signalling, there is also a wealth of literature linking various R-loop regulatory proteins to elevated immune signalling that has yet to be linked to their roles at Rloops.
The RNA/DNA helicase SETX is a well-studied regulator of R-loop homeostasis which functions by promoting their resolution.As previously discussed, SETX mutant cells display aberrant accumulation of Rloops which have been linked to elevated immune signalling [8], however, earlier studies had already demonstrated a role for SETX in regulating anti-viral responses, with a hyper-activated inflammatory state observed in SETX deficient cells upon viral infection [57].Evidence also suggests that SETX mutations in amyotrophic lateral sclerosis 4 (ALS4) leads to elevated CD8 T cell activity [58].XPG and XPF work with SETX and are endonucleases that cleave ssDNA; thus, they have been implicated in the processing of the ssDNA component of R-loops.Loss of either gene has been linked to innate immune activation [8] which may be due to increased R-loop levels.In addition, elevated R-loops in XPG/F sufficient settings may result in the establishment of inflammatory genomic instability resulting from excessive ssDNA cleavage [59].
RNASEH1/2 and Top1 are also negative regulators of R-loops [60,61].RNASEH enzymes degrade the RNA portion of the DNA-RNA hybrid and as previously discussed, mutations in these genes can lead to autoimmune diseases.Mice harbouring RNaseH2 mutations were shown to significantly upregulate ISGs via cGAS/STING signalling [49] and it was further demonstrated that loss of RNaseH2 led to the accumulation of micronuclei structures containing damaged DNA that could elicit downstream immune signalling, also via cGAS/STING [62].Top1 relaxes supercoiling in dsDNA and its deregulation has also been linked to autoimmune disorders, furthermore, it has been implicated in the regulation of various immune cells including bone marrow derived dendritic cells, T cells and B cells [63].
Recent studies have heavily implicated DExD/H box helicases in Rloop metabolism and subsequent immune signalling regulation.In their roles as R-loop resolvers, DExD/H box helicases are typically negative regulators of downstream immune signalling as is seen by the action of DDX5 (p68) in RORγt + regulatory T cells, where it inhibits HIF1α mediated IL-10 expression via the disassembly of R-loops at the transcriptional start site of the HIF1α locus [64].Loss of DDX5 in these settings caused an accumulation of R-loops and induction of IL-10 expression, demonstrating that R-loops also have a role in the transcriptional regulation of immune responses.Beyond this role, DDX5 has been described as an immune suppressor in many settings, including inducing the expression of IL-1β in glioma cells [65] and the negative modulation of STING activity [66].
There are several other instances in the literature that suggest an underlying link between R-loop regulatory proteins/genes and immune responses, further strengthening the argument that R-loops are important mediators of immunity.

Implications for cancer
It is well documented that pathological R-loops and their uncontrolled regulators can have roles in tumour progression through effects on DNA integrity, and the inability or failure to resolve R-loops is an evident driver of cancer progression [67].However, the immunogenic nature of R-loops poses a new lens through which R-loop contributions to cancer can be viewed.
Given the previously discussed role for R-loops in the regulation of HSPC development, it is intriguing to consider that deregulation of Rloop homeostasis and subsequent immune activation may lead to haematological disorders.As previously stated, the DExD/H-box helicases are important mediators of R-loops and DDX41 has recently been gaining attention in both the R-loop and cancer research communities.Alongside cGAS and IFI16, DDX41 is classified as an upstream regulator of STING signalling [68][69][70] and a bone fide tumour suppressor that has been shown to associate with R-loops, promoting their resolution and the protection of genomic integrity [34,71].DDX41 is highly expressed in myeloid cells and, as well as other members of the DEAD/H-box family, has been identified as a driver of inherited and sporadic forms of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) [72][73][74].In an effort to map the human R-loop proximal proteome, Mosler et al., confirmed the association of DDX41 with R-loops.They demonstrated that DDX41 loss led to genomic instability resulting from the accumulation of pathogenic R-loops, as well as the induction of an inflammatory response which they propose is a driving factor in AML/MDS pathogenesis (Fig. 2A) [71].Recent studies have shown that Fig. 2. Contributions of immunogenic R-loop species to cancer.(A) DDX41 is a tumour suppressor gene involved in the resolution of R-loops.When DDX41 is lost or mutated in myeloid cells, there is an observed accumulation of R-loops that are associated with genomic instability and downstream inflammatory signalling which has been implicated in the onset of AML and MDS [71].(B) Paraspeckles are interchromatin complexes recently demonstrated to associate with, and resolve, R-loops.Loss of core paraspeckle components (Che1/NEAT1) has been found to induce R-loop accumulation in multiple myeloma models.This is hypothesised to be the source of elevated DNA-RNA hybrids in patient samples and the driver of downstream inflammatory signalling mediated by cGAS/STING and RIG-I [76].(C) Adult T Cell leukaemia is an aggressive disease with poor prognosis, displaying constitutive activation of NF-kB signalling which was shown to drive R-loop accumulation [77].Various studies have linked R-loops to the induction of NF-kB signalling through the production of immunogenic by-products [9][10][11], raising the possibility that a feedback loop may exist that drives a sustained inflammatory phenotype.(D) Immunogenic R-loops have been implicated in various solid tumour types including breast, ovarian and colorectal.Loss of the tumour suppressor genes BRCA1 or SAMHD1 have been linked to elevated R-loops, genomic instability and the associated accumulating R-loop by-products which can induce downstream immune signalling via cGAS/STING.Chromatin associated cGAS has also been implicated in the slowing of replication forks which impedes R-loop formation, highlighting the multifaceted regulatory network in existence between R-loops and inflammatory signalling.Created with BioRender.com. the myocyte enhancing factor 2 (MEF2) transcription factor regulates IFN signalling activity and that the resulting inflammatory signalling observed upon MEF2 depletion results from increased R-loop levels and is dependent on DDX41 and ATR activity [75].
Accumulation of R-loop species can also lead to persistent activation of inflammatory responses in multiple myeloma (MM).Paraspeckles are irregularly shaped interchromatin complexes which are associated with the regulation of transcription, recently, they have also been implicated in R-loop homeostasis.Disruption of core paraspeckle components, NEAT1 or Che1, leads to an accumulation of transcriptional R-loops in MM cells.These cells also demonstrate activation of the NA sensors cGAS and RIG-I and concomitant downstream activation of NF-kB and IFN responses which was found to be dependent on accumulated R-loops (Fig. 2B) [76].Bruno et al., were able to demonstrate the clinical importance of their findings by showing that MM patients harbour a higher burden of DNA-RNA hybrids than healthy cells, and that these elevated levels correlated with high expression of IFN pathway components and poor prognosis.Interestingly, studies in adult T cell leukaemia have also uncovered a potential feedback loop in existence between R-loops and immune signalling in haematological malignancies, as NF-kB signalling has been found to induce R-loop accumulation (Fig. 2C) [77].These data alongside findings by Weinreb et al., [34] highlight the fine balance that exists between R-loops and inflammatory homeostasis in haematological settings.
There is also evidence that R-loop driven inflammation has a role in solid malignancies (Fig. 2D).Mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, lead to a significantly increased risk of developing breast and ovarian cancers.These genes have several reported roles including in DNA repair and replication amongst others, and they have a well-established role in the resolution of R-loops [78][79][80].As discussed, BRCA1 mutant cancer cells were shown to display accumulation of cytoplasmic R-loop by-products which induce innate immune responses [8].The AGS-associated gene SAMHD1 is also frequently linked to various malignancies including ovarian and colorectal cancers, as well as chronic lymphocytic leukaemia.Recent studies have proposed that SAMHD1 is a tumour suppressor that acts by resolving R-loops and that silencing of SAMHD1 can induce IFN-I production [53,81].The possibility of a feedback loop in solid cancers has also been raised as it was found that cGAS can inhibit the accumulation of R-loops in U2OS cells [82].
Together, these data strongly imply that dysregulated R-loops and subsequent inflammatory signalling are important pathological processes in various cancer settings which provide exciting opportunities for therapeutic development.Such an opportunity has recently been uncovered for the chemotherapeutic agents, topoisomerase I/II (TOPI/ II) poisons.A recent study has demonstrated that they can induce elevated micronuclei levels and inflammation, but also R-loop accumulation which has been linked to cGAS/STING mediated inflammatory signalling [83,84].It has also been suggested that TOPI may have a role in resolving R-loops [85] and so inhibition of this function may 'prime' cancers for improved response to rational combination therapies with immune checkpoint blockade following R-loop induced inflammatory signalling.It will be exciting to reveal how immunogenic R-loop species can be exploited in the treatment of cancer.

Outstanding questions & future direction
The role of R-loops in inducing innate immune responses represents an exciting emerging field, with several unanswered questions remaining.The importance of subcellular localisation in the detection and response to NAs is well documented, with structures such as micronuclei and endosomes known locales for concentrating signalling cascades.Therefore, it will be interesting to discover if the location of R-loop related species affects the resulting immune responses.It has been shown that R-loop by-products (ssDNA and DNA-RNA hybrids) can be transported to the cytoplasm, but it remains unknown if there are other mechanisms at play.Recent reports have described a role for R-loops in the stimulation of micronuclei formation [86] and so it will be interesting to uncover if R-loops can be processed in similar ways to other NA species; can they be sequestered in micronuclei?Must the micronuclei rupture?Do endosomal TLRs play a role in sensing R-loop products?Moreover, can nuclear R-loops be detected and responded to directly?
Another outstanding but important question is whether the type of Rloop species can affect the downstream immune response.To date, the detection of R-loop derived ssDNA and DNA-RNA hybrids has revealed similar downstream responses (Fig. 1), but this field is in its infancy and much remains unknown, including whether any of these by-products are more immunostimulatory than others.Emerging data implies that the context in which the R-loop species arise can lead to varying downstream responses and it would be rational to assume that DNA damage induced R-loops could influence downstream inflammation in a different manner to physiologically elevated co-transcriptional R-loops.Furthermore, one might assume that the sensors involved in sensing these different kinds of R-loop species may also differ.It will be interesting to note if the secondary structure of R-loops can induce immune responses, as has been reported for DNA G-Quadruplexes (G4) which are four-stranded secondary structures that can arise in guanine-rich sequences [87].The answers to these questions are undoubtedly being extensively studied already and will have far reaching implications in the study of many disease states.
As discussed throughout this review, a recurrent theme in the study of deregulated R-loops is that of autoimmune disorders with links to neurological dysfunction, raising the intriguing notion that the central nervous system (CNS) tissues may have a susceptibility to R-loop driven inflammation [7,88].For example, the DNA helicase SETX is an important mediator of R-loop resolution.Mutations in SETX can lead to the development of various diseases including amyotrophic lateral sclerosis 4 (ALS4) [89] and ataxia oculomotor apraxia type 2 (AOA2) [90].Both are autoimmune, neurodegenerative diseases that display devastating symptoms, and aberrant accumulation of R-loops has been reported in the cells of patients [8,91].Whilst these elevated R-loop levels have not, to the best of our knowledge, been unequivocally linked to the elevated immune responses seen in these diseases, the overwhelming body of evidence linking the two seem to predict that this is likely the case.Of note, studies in ALS4 have shown that SETX mutations lead to the expansion of CD8 T cells in the CNS which have heightened effector function against glioma, further suggestive of a sensitivity for R-loop driven inflammation in neuronal tissues [58].As previously discussed, different types of leukaemia also display a propensity for increased R-loop levels.Whether particular diseases are more vulnerable to R-loop linked immune signalling remains unknown but may hold important ramifications for future therapeutic development.
The overreaching aim of most biological studies is to develop cures for the myriad of diseases that plague humanity.As previously discussed, Chatzidoukaki et al. elegantly demonstrated the ability to attenuate R-loop driven inflammation in animal models of the premature aging disease, XFE, using relatively simple techniques.Crossley et al., also reported that digestion of cytoplasmic DNA-RNA hybrids in vitro led to decreased innate immune responses and apoptosis.These findings and the data discussed throughout this review highlight the vast potential that a deep understanding and appreciation of R-loop related immune responses holds for the development of treatments for a wide range of diseases, from cancer to progeria to autoimmune disorders.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.R-loops and their by-products as immunogenic species.(A) R-loops are three-stranded nucleic acid structures consisting of a single stranded DNA (ssDNA) and a DNA-RNA hybrid.(B) Processing and/or deregulation of R-loops homeostasis can lead to the formation and cytoplasmic release of R-loop by-products.(Left) Loss/mutation of SETX1 or BRCA1 can lead to genome wide R-loop accumulation.These R-loops are processed by XPG & XPF to form DNA-RNA hybrids which are transported to the cytoplasm and sensed by the PRRs cGAS & TLR3 [8].(Middle) Unresolved DNA damage, resulting from an inability to repair DNA (e.g.Ercc1 loss)or from natural aging, leads to the accumulation of R-loops and subsequently, ssDNA fragments, which are actively transported into the cytoplasm in an RPA-dependent manner.Cytoplasmic ssDNA is sensed by an unconfirmed sensor (*thought to be cGAS based on STING activation) and leads to the nuclear translocation of NF-kB and the activation of downstream inflammatory signalling[9].(Right) Aicardi-Goutières Syndrome (AGS) is a neuroinflammatory disease with features mimicking congenital viral infection.Mutations in RNASEH2 are detectable in > 50% of cases and have been found to cause the build-up of co-transcriptional R-loops which are processed by XPG and XPF, as well as an increase in micronuclei formation.Whilst it is yet to be confirmed if R-loop specific by-products are contained in these micronuclei, or if other intermediary species exist (such as DNA-RNA hybrids) following processing, R-loop accumulation has been linked to activation of NLRP3 inflammasome and cGAS/STING signalling[10,11].(C) The detection and response to pathogenic R-loop by-products leads to the activation of downstream inflammatory signalling mediated by various transcription factors such as IRF3 and NF-kB.Created with BioRender.com.