Topoisomerases in the immune cell development and function

Abstract

DNA topoisomerases are complex enzymatic machines with extraordinary capacity to maintain DNA topology during torsion intensive steps of replication and transcription. Recently, topoisomerases have gained significant attention for their tissue-specific function and the vital role of topoisomerases in immune homeostasis and dysfunction is beginning to emerge. Topoisomerases have been implicated in various immunological disorders such as autoimmunity, B cell immunodeficiencies, and sepsis, underscoring their importance in immune regulation. However, much remains unknown about immunological underpinnings of topoisomerases, and deeper understanding of the role of topoisomerases in the immune system will be critical for yielding significant insights into the etiology of immunological disorders. In this review, we first discuss the recent literature highlighting the contribution of topoisomerases in the development of immune cells and we further provide an overview of their importance in immune cell responses.

Introduction

The cellular processes such as transcription and replication are epitomized by the unwinding of two strands of DNA. However, this unwinding of DNA strands can promote intertwining of DNA molecules leading to the torsional stress. Topoisomerases (TOP) are molecular machines designed by nature to resolve topological problems in DNA arising due to strand separation (1, 2). In 1971, the identification and extraction of Escherichia coli ω protein, capable of unwinding negative supercoiled covalent closed DNA by James C. Wang, marks the discovery of topoisomerases-“true magicians of the DNA world” as he called them (3, 4). This was followed by the discovery of a "DNA untwisting enzyme" by James Champoux in 1972 from the nuclear extracts of murine embryo cells that was capable of resolving both negative as well as positive supercoils (5). In 1976, another protein with ATP-dependent DNA topoisomerase activity was discovered in E. coli and this enzyme was termed DNA gyrase (6). Eventually, the topoisomerases were divided into two categories: type I and type II topoisomerases with ω protein isolated by James C. Wang and "DNA untwisting enzyme" isolated by James Champoux being type I, and DNA gyrase identified subsequently being type II.

The mammalian genome carries seven topoisomerase encoding genes: four that encode type I topoisomerases (TOP1, TOP1mt, TOP3A and TOP3B) and other three encoding type II topoisomerases (TOP2A and TOP2B and SPO11) (4, 7, 8). Enzymatic activity of topoisomerases involves a highly reversible transesterification reaction through active site tyrosine residue resulting in the formation of an enzyme-DNA intermediate, referred as cleavage complex. Type I topoisomerases unwind the DNA by introducing single strand nick to allow another strand to pass through the gap. This enzymatic reaction does not require ATP and type I topoisomerases utilize the energy stored in torsional stress of supercoiled DNA. The type I topoisomerases are further divided into two subgroups, type IA and IB, depending on whether the enzyme is covalently linked to 5’ (type IA) or 3’ end (type IB) of the nicked DNA. TOP1 is a type IB enzyme that recognizes double stranded DNA and relaxes both positive and negative supercoils by a mechanism termed as ‘controlled rotation’ (9). TOP1mt is another member of class type IB and is known to maintain the integrity and topology of mitochondrial DNA. TOP1 inhibitors such as camptothecin and its derivatives, topotecan and irinotecan, stabilize TOP1 cleavage complex (TOP1cc) and block the religation step in reversible manner (10, 11). TOP3A and TOP3B, the two members of type IA family, facilitate the relaxation of negatively supercoiled but not positively supercoiled DNA (Fig. 1A) (7, 12).

Figure 1. Mammalian topoisomerases and the immune system.

(A) The classification of mammalian DNA topoisomerases is shown. Topoisomerases resolve the torsional strain generated during replication and transcription by cleaving either single or both strands of DNA and forming an intermediate cleavage complex. Based on the number of strands cleaved, they can be classified into type I and type II topoisomerases. Type I topoisomerases (TOP1, TOP1mt, TOP3A and TOP3B) do not require ATP and utilize the energy stored in torsional strain to either pass one strand through the other (type IA) or swivel around a strand (type IB). Type II topoisomerases (TOP2A, TOP2B and SPO11) are ATP-dependent enzymes. They create a transient break in one DNA double helix and pass another DNA double helix through it to relax the supercoiling. (B) The phenotype of mice models carrying genetic deletion of topoisomerases’ is shown. The embryos of Top3a knock-out mice are resorbed before 7.5-dpc. Mice without TOP3B have shorter lifespan and are infertile. Older cohorts of Top3b-null mice possess high level of autoantibodies. Homozygous disruption of Top1 is lethal and the development of Top1 knock-out embryos fails between 4- and 16-cell stages. Unlike Top1 knock-out, mice with germline deletion of Top1mt are viable and fertile. Top2a deletion results in termination of mice embryo development at the 4- or 8-cell stage. Top2b disrupted mice have impaired motor axon growth resulting in perinatal death of pups. Spo11-null mice are infertile due to apoptosis in spermatocytes. Panels (C-E) highlight the role of topoisomerases in T cell development and maturation. (C) Loss-of-function of TOP3A in zebrafish leads to selective defects in T cell development. Two mutations, HI064 and WW20/12, in Top3a impact thymopoiesis in zebrafish, with normal development of T cell progenitors but reduced number of thymocytes. (D) TOP1 and TOP2A are cardinal partners of AIRE with TOP1 localizing at super-enhancers while TOP2A at the promoter of AIRE-induced genes in medullary thymic epithelial cells. Inhibition of either TOP1 or TOP2A in mice leads to manifestation of autoimmunity. (E) While mice with Top3b deletion appear to be healthy, they develop autoimmunity with the progression of age. Panels (F-G) showcase the role of topoisomerases in B cell development and function. (F) The genetic basis of two human primary B cell immunodeficiencies, Hoffman syndrome and BILU, is the mutations in the TOPRIM domain of TOP2B. Hoffman syndrome and BILU are characterised by reduced B cell numbers and antibodies in circulation. On a similar line, deletion of Top2b in mice leads to perturbations in B cell compartment, while T cells remain largely unaffected. (G) In contrast to TOP2B, TOP1 plays a major role in B cell responses. Dynamic regulation of TOP1 is crucial for AID-dependent CSR and SHM. On stimulation of B cells, reduced levels of TOP1 either through haploinsufficiency or siRNA-mediated silencing lead to enhanced CSR and SHM. (H) TOP2B plays a critical role in NK cell development. Hoffman syndrome and BILU patients with mutations in Top2b and mice with heterozygous Top2b allele display reduced frequency of NK cells. TOP2B dysfunction in NK cells leads to lower transcript levels of Nfil3, Ets1, Id2, the key transcription factors for NK cell development.

TOP, topoisomerase; dpc, days post coitum; BILU, B cell immunodeficiency, limb anomalies and urogenital malformations; AIRE, autoimmune regulator; AID, activation induced cytidine deaminase; CSR, class switch recombination; SHM, somatic hyper mutation; NK, natural killer.

In contrast to type I topoisomerases, type II topoisomerases only form 5’ intermediates with DNA and require high energy cofactor as well as magnesium to catalyze topological changes in DNA. Type II topoisomerases manifest passage of one DNA double helix through a transient enzyme-bridged break in another to manage DNA tangles and to relax positively or negatively supercoiled DNA (7, 13). Vertebrates and other multicellular eukaryotes have two bonafide type II topoisomerase paralogs, TOP2A and TOP2B, often referred as TOP2 collectively, that share ~79% sequence identity in their catalytic core while C-terminal domain (CTD) of the two enzymes differs significantly (~31% identity) (14, 15). It is believed that CTD of type II topoisomerases plays a role in sensing the geometry of DNA substrates with TOP2A preferentially relaxing positively supercoils while display of no such preference by TOP2B (14). Similar to camptothecin, drugs such as etoposide, teniposide, and anthracycline family drugs including epirubicin, doxorubicin, and daunorubicin function by stabilizing an intermediate wherein DNA is covalently linked to the enzyme i.e. TOP2 cleavage complex (TOP2cc) (15, 16). Eukaryotic genome also encodes a third atypical type II topoisomerase or more precisely topoisomerase-like protein, SPO11, that is germ-cell specific and is active during meiosis (Fig. 1A) (17).

The dysfunction of topoisomerases in various eukaryotic model organisms has provided important insights into the essential nature of these enzymes and their exquisite role in tissue homeostasis. For example, Top1 is dispensable in unicellular eukaryotes like yeast (18–20); however, it is essential for early development in multicellular eukaryotes including fruit flies and mice (21, 22). On the contrary, Top1mt knock-out mice are viable but display a phenotype under stress conditions (23–26). Similar to Top1, deletion of Top3a gene leads to embryonic lethality in mice (27), while Top3b knock-out mice do not show developmental defects but display aneuploidy in the spermatocytes and develop autoimmunity (28–30). Similarly, deletion of Top2a gene results in lethality in mice during embryonic development, while Top2b-deficient murine embryos display neural and neuromuscular abnormalities resulting in perinatal death (31, 32). Spo11-null mice are viable but carry defective spermatocytes leading to infertility (Fig. 1B) (17, 33).

It is becoming increasingly clear that the purview of topoisomerases extends beyond the basic enzymatic function, and they have a role in regulation of tissue homeostasis. However, owing to pleiotropic functions of topoisomerases and embryonic or perinatal death of gene-deficient animal models, it has been challenging to parse the tissue-specific function of topoisomerases. Studies on non-lethal mutations in topoisomerase genes, mice carrying a conditional topoisomerase allele deletion and pharmacological intervention of topoisomerase enzymes have shed some light on the tissue-specific role of these enzymes. Indeed, recent findings have indicated that both type I and II topoisomerases play a key role in post-mitotic neurons and immune cells (8, 29, 32, 34–39). In this review, we focus on the immune system-specific requirements of topoisomerases and their importance in immunologic disorders. First, we highlight the importance of topoisomerases in the development of diverse immune cell populations and second, we discuss the role of topoisomerases in immune responses.

Role of topoisomerases in adaptive immune system

T cells

An initial indication of the importance of topoisomerase function in the immune system was the observation that T cell development is uniquely sensitive to loss-of-function of TOP3A in zebrafish. Boehm and colleagues, while performing a forward genetic screen to identify recessive mutants with thymopoiesis defects in zebrafish embryos, identified two Top3a alleles, HI064 and WW20/12, with abnormalities in thymopoiesis (Fig. 1C). HI064 is a null Top3a allele as it contains a nonsense mutation and translated protein is predicted to be a truncated version of wild-type (WT) TOP3A which lacks a significant portion of topoisomerase IA homology domain containing the catalytic tyrosine residue and three zinc fingers. WW20/12 contains a missense mutation in the topoisomerase IA homology domain replacing a hydrophobic residue with a hydrophilic and polar residue very close to three highly conserved amino acid residues critical for contact with the single-stranded DNA substrate. The zebrafish with either HI064 or WW20/12 allele displays indistinguishable thymopoiesis abnormality phenotype suggesting the missense mutation in WW20/12 allele probably produces non-functional TOP3A. Interestingly, in Top3a mutant embryos, analysis of neuronal development in the eye and brain showed little perturbation, indicating that loss of TOP3A did not strongly affect early neurogenesis. However, the mutant zebrafish embryos displayed compaction of epithelial layer in thymus which is critical for maturation of thymocytes. Most importantly, although lymphocyte progenitors develop normally and have normal thymic migration in the Top3a mutant embryos, deletion of Top3a significantly impacted the number of thymocytes (40).

TOP1 and TOP2A are also involved in the development of mature T cells through their interaction with a thymic regulator of immunological tolerance, transcription factor-Autoimmune Regulator (AIRE). AIRE drives the expression of a battery of peripheral-tissue antigens in medullary thymic epithelial cells (mTECs), and thereby regulates the deletion of self-reactive T cells during development. TOP1 and TOP2A were shown to serve as cardinal partners for AIRE and these two topoisomerases regulated the AIRE-directed transcription program. Interestingly, two classes of topoisomerases had differential and non-redundant functions in AIRE-induced gene expression, where TOP1 primarily functioned with AIRE at super-enhancers, while TOP2A interacted with AIRE at the promoter of AIRE-induced genes. The inoculation of mice either with topotecan (TOP1 inhibitor) or etoposide (TOP2 inhibitor) was able to establish autoimmune manifestations similar to those seen in AIRE-less disease further supporting the role of two groups of topoisomerases in the development of T cells (Fig. 1D) (41, 42).

Interestingly, though targeted mutation of Top1 and Top2a leads to embryonic lethality (22, 31), mice lacking Top3b gene develop to maturity with no apparent defects (28). However, Top3b-null mice develop autoimmunity as they age with elevated levels of circulating autoantibodies, a phenotype peculiar of AIRE-less disease (29). Although incremental levels of autoantibodies in Top3b-null mice were attributed to apoptotic cells in thymus, an AIRE-dependent role of TOP3B in T cell development and maturation can’t be ruled out (Fig. 1E).

B cells

A clear demonstration of the need for topoisomerase activity in B cell development has come from inherited human syndromes. Studies aiming to identify the genetic defects in the patients with reduced or absent B cells and hypogammaglobulinemia showed the role of TOP2B in B cell development. Papapietro et. al. and Broderick et. al. independently identified dominant loss-of-function mutations in Top2b in five families. They demonstrated that the mutations in Top2b cause syndromic B cell immunodeficiency namely, Hoffman syndrome and BILU (B cell immunodeficiency, Limb anomalies, and Urogenital malformations), and suggested that these mutations underlie the defects in B cell development and B cell activation in response to antigen stimulation (43, 44). Along similar lines, another clinical study recently reported absolute B cell deficiency in a 13-year-old patient with craniofacial and limb abnormalities and the patient carried Top2b mutations (45). Patients with Top2b mutations have a distinct lack of CD19⁺ B cells in the bone marrow suggesting a block in early B cell development and making this immunodeficiency different from other B cell immunodeficiencies (Fig. 1F). However, these patients have a minor fraction of B cells and detectable levels of immunoglobulins circulating in the peripheral blood suggesting that B cells in the patients are not entirely lost and hinting towards a leaky block in the development of B cells. Most importantly, the defects in Top2b led to specific inhibition of B cell development with peripheral T cells remaining unaffected despite the fact that both T and B cells originate from common lymphocyte precursor. Interestingly, a significant number of patients with Hoffman syndrome exhibited reduced number of natural killer (NK) cells in blood and we will discuss this observation in a later section of this review. Most importantly, the immunodeficiency in patients with Top2b mutations was restricted to lymphocyte lineage with myeloid lineage not being affected (44, 45). Based on these observations, International Union of Immunological Societies (IUIS)’s expert committee for Inborn Errors of Immunity (IEI) (https://iuis.org/committees/iei/) has recently included mutations in Top2b gene as one of the genetic cause for antibody deficiency in humans, and Top2b can be a candidate gene for screening mutations in patients demonstrating B cell deficiency (46).

The mutations in patients with Hoffman syndrome and BILU syndrome were reported at conserved residues in the TOPRIM (topoisomerase-primase) domain of TOP2B. The TOPRIM domain of TOP2B is part of the DNA gate that catalyzes DNA cleavage and religation and is essential for catalytic activity of TOP2B. Interestingly, the Top2b mutations led to lower intrinsic enzymatic activity and reduced stability of the protein, potentially precipitating Top2b-null phenotype in the patients. Most importantly, these patient mutations create the dominant negative form of TOP2B which contributes to disease phenotype rather than Top2b haploinsufficiency (43).

Interestingly, while deletion of Top2b in murine B cell lineage affected B cell numbers in spleen and bone marrow, the extent of developmental deficiency was milder when compared to patients (Fig. 1F). The stronger effect on development of B cells in patients can be ascribed to the dominant-negative effect of the mutation on the functioning of TOP2B derived from the WT allele.

While TOP2B plays a key role in development of B cells, TOP1 has been demonstrated to play a role in antigen-dependent antibody maturation in B cells. On antigen stimulation, the immunoglobulin locus undergoes two distinct genetic modifications to generate antibody diversity: class switch recombination (CSR) and somatic hypermutation (SHM). Activation induced cytidine deaminase (AID) is an enzyme that mediates CSR and SHM by promoting DNA cleavage in the switch and variable regions of the immunoglobulin (Ig) locus respectively (47). It was shown that dynamic regulation of TOP1 expression is responsible for AID-dependent CSR and SHM and increased expression of AID correlated with reduced TOP1 levels in the cell (48, 49). siRNA-mediated gene silencing of Top1 promoted AID-induced DNA cleavage, CSR and SHM. Similarly, Top1 haploinsufficiency in mice led to increased frequency of SHM in B cells. Interestingly, SHM augmentation was dependent on transcription as inhibition of transcription had negative impact on SHM, even in cells with Top1 knock-down (KD). It was proposed that reduced expression of TOP1 protein promotes transcription-induced non-B DNA structure formation which offers sites for irreversible cleavage by TOP1, and this DNA cleavage augments CSR and SHM. Interestingly, poisoning of TOP1 by camptothecin, which stabilizes DNA-TOP1 cleavage complex, inhibits both CSR and SHM, suggesting that the clearance of TOP1-DNA complexes is also critical for CSR and SHM (Fig. 1G).

Similar observations were made in a mouse B-lymphocytic leukemia cell-line P388/CPT45 that displays aberrant expression of Ig on cell surface and it was attributed to insufficient expression of TOP1 (49, 50). P388/CPT45 cells do not express endogenous AID, but it was observed that exogenous expression of AID in this TOP1-insufficient line led to increased SHM frequency, while overexpression of human TOP1 resulted in reduced SHM. Proteomic analysis of TOP1-associated proteins in this B cell line pointed towards key role of SMARCA4, an ATP-dependent chromatin remodeler, in recruitment of TOP1 on chromatin as B cell line with Smarca4-KD phenocopied the effects of Top1-KD on SHM (51). These results further highlight regulatory role of TOP1 protein levels in the process of antibody-maturation in B cells.

NK cells

NK cells are at the cross-roads of innate and adaptive immunity (52). Akin to B cells, the evidence for role of topoisomerases in NK cell development and function has come from research on patients with immunodeficiency. Two recent studies reported that a significant proportion of patients suffering with Hoffman syndrome and BILU syndrome had lower frequencies of NK cells and these patients carried loss-of-function mutations in Top2b gene (Fig. 1H) (43, 53). However, unlike B cells, the effect of Top2b mutations on NK cells development was much more variegated with a fraction of patients having normal NK cell frequencies, and no patient displaying complete loss of NK cells. Most importantly, defects in NK cells were much more common in the patients with Hoffman syndrome in comparison to the patients with BILU syndrome, further highlighting the differential role of specific TOP2B residues in NK cell development and function. Interestingly, the study reported 3 patients with BILU syndrome, and all 3 patients carried A485P mutation in TOP2B; however, only one of the patients displayed significantly lower frequencies of NK cells. This observation suggests the contribution of additional factors in NK cell defects observed in patients with BILU syndrome.

Surprisingly, induced pluripotent stem cells (iPSCs) from healthy donors and patients with Hoffman syndrome displayed similar NK cell differentiation potential in vitro. However, iPSC-derived NK cells from Hoffman syndrome patients had impaired cell-mediated cytotoxicity towards cancer cells. These results suggest that Top2b mutant iPSCs have the potential to differentiate into NK cells, but the differentiated cells lack the functional attributes of NK cells (53).

As observed in patients with Hoffman syndrome, mice carrying mutation in Top2b gene showed fewer mature NK cells compared to WT counterparts, and these cells had limited cytotoxic killing activity towards cancer cells. Interestingly, TOP2B controlled the expression of transcription factors Nfil3, Ets1, and Id2 that are known to regulate the NK cell development (Fig. 1H) (53). Overall, these results highlight previously unknown role of TOP2B in NK cell differentiation and function.

Role of topoisomerases in innate immune system

Although the role of topoisomerases in development of innate immune cells still needs to be explored, topoisomerases are known to play key roles in the functioning of innate immune cells. Various lines of evidence suggest that topoisomerases regulate innate immune responses. However, there is significant granularity in their precise role in the regulation of immune responses across innate immune cell types.

The use of pharmacological inhibitors to delineate the role of topoisomerases in these cells has been highly productive and has paved way for discovery of their novel roles in the functionality of innate immune cells. Analysis of antineoplastic drugs, including a TOP2 poison doxorubicin, was found to have marked effect on the maturation of human dendritic cells (DCs). Inhibition of TOP2 by doxorubicin, at ultra-low non-cytotoxic concentrations (10nM), led to a marked increase in the expression of costimulatory molecules such as CD40 and CD83 on human DCs (54). Interestingly, the ability of doxorubicin to induce maturation of human DCs did not enhance its T cell activation potential in mixed lymphocyte reaction (MLR) assay (54). On similar lines, TOP1 inhibitor camptothecin and its analogs topotecan and irinotecan, induced maturation of murine bone marrow derived DCs (BMDCs) with significantly elevated surface expression of MHCII, CD40, and CD80 on these cells. Interestingly, as observed with TOP2 inhibitors, TOP1 inhibitors did not augment the capacity of BMDCs to activate naive T cells in MLR assay (55). These results bring forward an important point that TOP1 and TOP2 inhibitors induce partial maturation of DCs with the functional hallmark of DC maturation – T cell activation potential-not being affected.

On the other end of the spectrum, chemical inhibition of TOP1 suppressed the pro-inflammatory immune response against pathogenic infections at cellular as well as organismal level. Rialdi et. al. performed a chemical screen for innate immune system-intrinsic regulators of the transcriptional response to pathogens and observed an inhibitory activity of TOP1 inhibitor, camptothecin, on the expression of pro-inflammatory genes. Notably, inhibition of TOP1 activity compromised the inflammatory immune response against a variety of viral and bacterial pathogens, and their products. Most importantly, the repression of pro-inflammatory gene expression was not due to camptothecin-mediated stabilization of TOP1cc or induced cell damage as siRNA-mediated depletion of Top1 phenocopied the attributes of TOP1 inhibition, and the effect of TOP1 inhibition on inflammatory genes was reversed by drug washout. Interestingly, in vivo administration of camptothecin alleviated lethality in a murine model of sepsis highlighting therapeutic potential of TOP1 inhibition for diseases characterized by exacerbated innate immune responses (56). In fact, a recent study by Ho et. al. proposed the need of evaluating the efficacy of TOP1 inhibitor, topotecan, for severe coronavirus disease 2019 (COVID-19) in humans as therapeutic treatment with two doses of topotecan in animal models suppressed lethal inflammation induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (57). Similarly, camptothecin was shown to inhibit inflammatory cytokine production by lipopolysaccharide (LPS) or interferon (IFN)-γ-stimulated microglial cells, and TOP1 inhibition by topotecan mitigated neuroinflammation in mice as well as ameliorated disease pathology in murine model for experimental autoimmune encephalomyelitis (EAE) (58). On the contrary, deletion of TOP1 in excitatory neurons led to intense IBA1 immunostaining in the somatosensory cortex indicative of extensive neuroinflammation in these conditional Top1 knock-out mice. These results suggest that TOP1 may control neuronal health to limit neuroinflammation (36). It is possible that TOP1 plays a different role in steady-state vs inflammatory conditions and more studies towards this direction are needed to fully comprehend the function of TOP1 in health and immune-mediated diseases.

In contrast to TOP1, the role of type II topoisomerases - TOP2A and TOP2B - in innate immune responses has been debatable. The use of different infection and cellular models may explain the discrepancy in results across studies. Rialdi et. al. reported that loss-of-function of TOP2A and TOP2B by siRNA-mediated knock-down or by chemical inhibition does not phenocopy the effect of TOP1 inhibition on the expression of pro-inflammatory genes upon viral infection in A549 cells (56). On the contrary, type II topoisomerases have been found to be important for pro-inflammatory cytokine production by macrophages as well as for innate immune responses in murine models. Etoposide treatment led to a decrease of LPS or IFN-γ-induced tumor necrosis factor (TNF)-α production by IC-21 cells (macrophage cell line). However, it was also observed that perturbation in the expression of interleukin (IL)-6 upon treatment with etoposide varied with kind of stimulation. While etoposide reduced LPS-stimulated production of IL-6, it augmented IFN-γ-induced expression of IL-6. These results highlight differential role of type II topoisomerases in the transcriptional circuitry established upon LPS or IFN-γ stimulation of macrophages. Interestingly, inhibition of type II topoisomerases with etoposide in NMRI mice led to an increase in numbers of monocytes and granulocytes but when splenocytes from these etoposide-treated mice were stimulated with TSST-I and ConA, they displayed reduced capacity to produce inflammatory cytokines (IL-6 and TNF-α) (59). Similarly, anthracycline family drugs including epirubicin, doxorubicin, and daunorubicin that are known to inhibit type II topoisomerases alleviated pro-inflammatory cytokine production from human monocytic cell-line THP1 in response to E. coli challenge. A similar disease ameliorating effect of epirubicin was observed in cecal ligation and puncture (CLP) model of sepsis (60).

Another interesting evidence for the role of type II topoisomerase TOP2A in innate inflammatory responses comes from a murine model for sepsis-induced acute lung injury (ALI). Mice with ALI had elevated expression of TOP2A, while levels of miR-125b-5p were downregulated. It was demonstrated that miR-125b-5p acts as an inhibitor of TOP2A expression. Interestingly, augmented miR-125b-5p expression led to reduction in levels of inflammatory cytokines such as TNF-α, IL-1β, IL-6 and improved lung pathology in mice model with ALI. However, TOP2A overexpression deflated miR-125b-5p-mediated health benefits, suggestive of critical role of TOP2A in inflammatory disease attributes of sepsis (61).

Furthermore, TOP1 and TOP2 are involved in the maintenance of macrophage identity and functioning of these cells, through their interaction with Speckled Protein 140 (SP140). SP140 is an immune restricted speckled protein with major activity in macrophages and mature B cells, and mutations in SP140 have been implicated in Crohn’s disease (62–65). Perturbation of interaction between SP140 and TOP1/TOP2 led to de-repression of topoisomerase activity at developmentally silenced genes in macrophages. Interestingly, functional dysregulation of macrophages due to loss of SP140 could be rescued by inhibition of TOP1 and TOP2, hinting towards the role of topoisomerases in maintaining the fidelity of macrophage cell lineage and promoting innate immune responses (66). Altogether, these observations bring forward the potential of TOP1 and TOP2 inhibition as an effective host-directed therapy to limit lethal systemic inflammation during broad spectrum pathogenic infections as well as other inflammatory disorders.

TOP1 and TOP2 have also been implicated in invasive cytosolic DNA sensing - an integral component of innate immunity. Zhao et. al. and Wang et. al. independently demonstrated the key role of TOP1 and type II topoisomerases in cyclic cGMP-AMP synthase (cGAS) mediated sensing of cytoplasmic chromatin. cGAS is a pattern recognition receptor that recognizes cytoplasmic chromatin fragments during senescence to mount an inflammatory immune response. It was shown that stabilization of TOP1cc or TOP2cc in tumor cells enhanced the binding of cGAS to dsDNA and induced innate immune signaling to promote expression of chemokines such as CCL5 and CXCL10 and type I interferons including IFN-β (67, 68). This in turn led to activation of DCs and T cell recruitment at the site of tumor. Inhibition of type II topoisomerases by teniposide also promoted upregulation of the antigen presentation machinery on tumor cells including enhanced expression of MHCI, MHCII, β2m, TAP1, and TAP2 that further promoted T cell activation against tumor and helped in tumor clearance (67). These results highlight indirect role of topoisomerase inhibitors in tumor regression via stimulatory activity towards innate immune sensing, enhancing antigen presentation capabilities and setting up the events for tumor infiltration of other immune cells.

In summary, both type I and II topoisomerases play diverse roles in innate immune responses. On one hand, topoisomerase inhibitors drive partial maturation of DCs, while on the other hand, inhibition of topoisomerases restrains inflammatory cytokine production in various cellular and animal models. However, current studies also highlight a challenge- and gene-dependent role of topoisomerases during inflammatory response. This is exemplified by the differences in perturbation of IL-6 levels upon inhibition of type II topoisomerases when cells are challenged with either LPS or IFN-γ. Similarly, TOP1 also appears to regulate innate immune responses differently in steady-state vs inflammatory conditions. Table 1 summarizes various roles of topoisomerases in the functioning of innate immune system.

Table 1. A tabular compilation of topoisomerase dysfunction phenotype in the innate immune system.

Molecule	Mode of disruption	Mice strain/Cell type	Type of stimulation	Outcome	References
TOP1/TOP2	Doxorubicin, Camptothecin, Topotecan, Irinotecan	Dendritic cells	No stimulus	Enhanced DC maturation, did not alter the T cell activation potential of DCs.	(54, 55)
TOP1	Camptothecin, Topotecan, siRNA	A549/RAW264.7 cell lines	Influenza virus, Lipopolysaccharide (LPS), SARS-CoV-2	Reduced expression of pro-inflammatory genes.	(56, 57)
TOP1	Camptothecin, Topotecan	Microglia cells	LPS/Interferon γ (IFNγ)	Decreased production of inflammatory cytokines.	(58)
TOP1	Knock-out	Top1 cKO mice	No stimulus	Increased neuro-inflammation and neuronal death.	(36)
TOP2	Etoposide	IC-21 macrophage cell line	LPS/IFNγ	Decreased production of IL-6 upon LPS treatment whereas increased production of IL-6 upon IFNγ challenge.	(59)
TOP2	Etoposide	NMRI mice	TSST-1 and ConA	Splenocytes exhibited reduced potential to produce cytokines.	(59)
TOP2	Epirubicin, Doxorubicin, Daunorubicin	THP-1 cell line	E. coli	Alleviated pro-inflammatory cytokine production.	(60)
TOP2	Epirubicin	Cecal ligation and puncture (CLP) model of sepsis	Polymicrobial infection	Confers disease tolerance.	(60)
TOP2A	miR-125b-5p agomir	CLP model of sepsis	No stimulus	Suppressed inflammatory response. Alleviated lung pathology.	(61)
TOP2	Teniposide	Tumor cells	No stimulus	Induced cytokine production, DC activation and T cell recruitment.	(67)

Conclusions

Since their discovery in 1971, topoisomerases have garnered tremendous interest and evidence gathered over the past two decades underscores the tissue-specific roles of these enzymes. In this review, we aimed to bring attention to the possible involvement of these enzymes in the development and function of immune cells. Topoisomerases are associated with immunodeficiencies, autoimmune disorders and inflammatory diseases (29, 40, 56, 58, 61), highlighting their unappreciated role in the maintenance of immune homeostasis and the regulation of immune responses. We have just started to learn about their role in adaptive immune system with studies pointing towards their function in thymopoiesis and the development of B and NK cells (40, 43, 44, 53). However, insights into their role in the development of innate immune cells are still lacking. Nonetheless, we have begun to appreciate the role of topoisomerases in innate immune responses, and these enzymes may regulate transcriptional programing of innate immune cells such as macrophages and DCs during pathogenic challenge (54–56).

Though the immunological underpinnings of topoisomerases are becoming increasingly evident; yet, we have a lot to learn. An important aspect that is largely unexplored is the mechanism of topoisomerases in the regulation of immune cell differentiation and function. Existing reports on the role of topoisomerases in neurons indicate towards a gene length dependent activity (38). Similar observations were made by Broderick et. al. for TOP2B in B cells, where ablation of Top2b gene in murine B cells led to reduced expression of key B cell specific transcription factors that are notably longer (Ebf, 390 kb; Pax5 178 kb; and Foxo1, 85 kb) (43). Interestingly, recent studies also reveal a dynamic interplay between topoisomerases and chromatin in immune cells. Rialdi et. al. found that 75% of TOP1-dependent immune response genes required switch/sucrose nonfermenter (SWI/SNF)-dependent nucleosome remodeling for transcriptional activation (56). In parallel, TOP1 was found to interact with SMARCA4 (also referred as BRG1) which is the ATPase subunit of SWI/SNF complex in B cells (51). We previously demonstrated the occupancy of TOP1 at H3K27ac-marked super-enhancers in mTECs (42). These observations are suggestive of an epigenetic layer in the molecular mechanism of topoisomerases. It is possible that there is a dysregulation of cross-talk between topoisomerases and chromatin remodeling machinery in immunological disorders and this axis can be harnessed to design new putative therapies for these diseases.

Another important mechanistic facet of topoisomerases is their role in genome organization. TOP2B was recently demonstrated to interact with chromatin architectural proteins CCCTC-binding factor (CTCF) and the subunits of cohesin complex that regulate enhancer-promoter interaction and the formation of chromatin loops (69). Interestingly, chromatin loop anchors bound by CTCF and cohesin are vulnerable to TOP2B-mediated DNA double stranded breaks in murine B cells and these DNA breaks may help in clearing up intertwined DNA that builds up during chromatin loop formation (70). Thus, TOP2B might control the genome organization and this regulatory axis may drive the cellular phenotypes observed in immune cells. Since the toolkit of topoisomerases is expanding, it will be important to know whether different tools are used across diverse immune cells and this variegation can be utilized to therapeutically target topoisomerases in specific immune cell type.

The versatility of topoisomerases and their ease for targeting through drugs makes them a viable molecule for translational research. Several topoisomerase poisons are used in cancer chemotherapy. Topoisomerases have also recently gained traction as ‘go to’ target molecule for diseases like rheumatoid arthritis, SARS-CoV-2 induced lethality and sepsis. Thus, understanding the role of topoisomerases in immune cell development as well as immune responses becomes essential to design drug regimens with least possible adverse consequences. This knowledge will also aid in developing better therapeutic regimens for regression of cancers as topoisomerase-directed chemotherapy may impact gene expression programs in immune cell populations and can have indirect impact on cancer. Overall, further insights into the immunological role of topoisomerases will likely have a significant impact on the well-being of human race.

Abbreviations used in the manuscript

AID

activation induced cytidine deaminase

AIRE

autoimmune regulator

ALI

acute lung injury

BILU

B cell immunodeficiency limb anomalies and urogenital malformations

BMDCs

bone marrow derived dendritic cells

cGAS

cyclic cGMP-AMP synthase

CLP

cecal ligation and puncture

CSR

class switch recombination

CTD

C-terminal domain

DCs

dendritic cells

IFN

Interferon

Ig

immunoglobulin

IL

interleukin

iPSCs

induced pluripotent stem cells

KD

knock-down

LPS

lipopolysaccharide

MLR

mixed lymphocyte reaction

mTECs

medullary thymic epithelial cells

NK

natural killer

SHM

somatic hypermutation

SP140

speckled protein 140

TNF

tumor necrosis factor

TOP

topoisomerase

TOP1cc

TOP1 cleavage complex

TOP2cc

TOP2 cleavage complex

WT

wild-type