The Three Prime Repair EXonuclease I (TREX1) is critical for degrading post-apoptosis DNA. Mice expressing catalytically inactive TREX1 (TREX1 D18N) develop lupus-like autoimmunity due to chronic sensing of undegraded TREX1 DNA substrates, production of the inflammatory cytokines, and the inappropriate activation of innate and adaptive immunity. This study aimed to investigate Thelper (Th) dysregulation in the TREX1 D18N model system as a potential mechanism for lupus-like autoimmunity. Comparison of immune cells in secondary lymphoid organs, spleen and peripheral lymph nodes (LNs) between TREX1 D18N mice and the TREX1 null mice revealed that the TREX1 D18N mice exhibit a Th1 bias. Additionally, the T-follicular helper cells (Tfh) and the germinal celter (GC) B cells were also elevated in the TREX1 D18N mice. Targeting Bcl6, a lineage-defining transcription factor for Tfh and GC B cells, with a commercially available Bcl6 inhibitor, FX1, attenuated Tfh, GC, and Th1 responses, and rescued TREX1 D18N mice from autoimmunity. The study presents Tfh and GC B-cell responses as potential targets in autoimmunity and that Bcl6 inhibitors may offer therapeutic approach in TREX1-associated or other lupus-like diseases.

Autoimmunity can result when cells fail to properly dispose of DNA. Mutations in the three-prime repair exonuclease 1 (TREX1) cause a spectrum of human autoimmune diseases resembling systemic lupus erythematosus. The cytosolic dsDNA sensor, cyclic GMP-AMP synthase (cGAS), and the stimulator of IFN genes (STING) are required for pathogenesis, but specific cells in which DNA sensing and subsequent type I IFN (IFN-I) production occur remain elusive. In this study, we demonstrate that TREX1 D18N catalytic deficiency causes dysregulated IFN-I signaling and autoimmunity in mice. Moreover, we show that bone marrow-derived cells drive this process. We identify both innate immune and, surprisingly, activated T cells as sources of pathological IFN-alpha production. These findings demonstrate that TREX1 enzymatic activity is crucial to prevent inappropriate DNA sensing and IFN-I production in immune cells, including normally low-level IFN-alpha-producing cells. These results expand our understanding of DNA sensing and innate immunity in T cells and may have relevance to the pathogenesis of human disease caused by TREX1 mutation.

Mutations in the 3′ ‐> 5′ exonuclease TREX1 are associated with a spectrum of autoimmune phenotypes in humans and mice. In those affected, pathology is driven by persisting DNA which would normally be degraded by functional TREX1 enzyme. This undegraded DNA stimulates the nucleic acid sensing pathway, cGAS‐ STING, whose subsequent signaling generates a type‐I interferon response. This type‐I interferon response ultimately results in the chronic immune system activation typical of those affected. TREX1 and the cGAS‐ STING pathway have also been implicated in the tumor microenvironment, where TREX1 is believed to degrade tumor‐derived DNA destined to trigger cGAS‐STING signaling. If this tumor‐derived DNA is not degraded, the stimulated cGAS‐STING pathway promotes IFN‐dependent antitumor immunity. Consequently, small molecule stimulators of the cGAS‐STING pathway have shown promise as immunotherapies in animal models. Similarly, mice devoid of TREX1 exonuclease activity exhibit T‐cell dependent resistance to tumor growth. Collectively, these findings indicate that stimulation of the cGAS‐STING pathway activates the immune system, and that TREX1 inactivation represents a novel strategy for this purpose. Furthermore, they demonstrate the immunotherapy potential of such approaches. Thus, we hypothesize that small molecule inhibitors of TREX1 will activate the cGAS‐STING pathway. Studies described here identify small molecule inhibitors of TREX1, determine their mechanism of inhibition, and directly test the hypothesis that they can activate the cGAS‐STING pathway in murine cells. Findings from these studies will generate new structural insights into how TREX1 exonuclease activity may be affected, and have already identified several candidate chemotypes for development into novel immunotherapies.Support or Funding InformationThis work was supported by the NIH, NIAID, NIGMS, Lupus Research Alliance, Wake Forest Innovations, and a Sandy Lee Cowgill Memorial Scholarship.D18N mice display T‐cell dependent tumor resistance.5×106 H31ml tumor cells were injected subcutaneously into WT and D18N mice, and tumor volume and survival tracked daily. Data in ‘B’ are separate study from ‘A’. For each study, eight to 16 mice were examined in two to four independent experiments. (A) D18N mice have decreased tumor growth in a mouse tumor model. (B) D18N model tumor‐resistance is T‐cell dependent. WT and D18N were treated with αCD8, αCD4, or isotype‐control antibodies. Arrows indicate D18N mice without treatment, or with CD8+ / CD4+ T‐cell depletion.Figure 1

Background Failure to properly dispose of self-DNA can inappropriately trigger anti-viral defense systems, leading to autoimmunity. Indeed, mutations in the DNA exonuclease TREX1 are causative for a spectrum of rare lupus-like autoimmune diseases in humans. These disorders involve triggering of the cytosolic dsDNA sensor cyclic GMP-AMP Synthase (cGAS) and the STimulator of INterferon Genes (STING), leading to chronic production of the anti-viral cytokine type I interferon (IFN-I) and the development of autoimmunity. Importantly, the exact cells in which the sensing of undegraded DNA and subsequent production of IFN-I occur remain unknown. Methods We generated a mouse expressing the catalytically inactive TREX1 D18N allele, which causes familial chilblain lupus in humans. We examined anti-viral gene expression and the phenotype of these mice to study the immunological effects of losing TREX1 activity. We performed bone marrow transplants to determine if autoimmune pathogenesis in this model was dependent on hematopoietic or non-hematopoietic cells. Finally, we measured expression of type I interferon is various purified cell populations to identify specific cellular producers contributing to autoimmune pathogenesis. Results In this study, we demonstrate that TREX1 catalytic inactivity induces IFN-I signaling and lupus-like autoimmunity in a mouse. Moreover, we show that TREX1 deficiency within bone marrow-derived cells causes IFN-I activation and the development of autoimmunity. We provide evidence of spontaneous IFN- production within both innate immune and T cells. T cell IFN-a expression was observed in all T cell populations, but was most enriched within naive T cells. We also demonstrate that D18N T cells express all components of the cGAS-STING pathways and generate IFN-I protein, both spontaneously and in response to small-molecule activation of STING.Abstract 284 Figure 1 TREX1 D18N T cells spontaneously generate IFN-a. (A) Kaplan-Meir survival curve demonstrating the absence of autoimmune mortality in TREX1 D18N IFNAR KO animals. (B) qRT-PCR measurement of IFN- gene expression in uninfected WT splenocytes, LCMV +splenocytes, purified LCMV +non innate immune cells, and purified LCMV +innate immune cells (data represents cells from three virally-challenged mice 48 hours after infection). (C) IFN- gene expression in splenocytes from unchallenged WT and D18N mice (26–30 mice per genotype) (D) IFN- gene expression in whole splenocytes and purified non-innate and innate immune cells from both WT and D18N mice (3 separations, 6–8 mice per genotype) (E) qRT-PCR measurement of IFN- expression or ISG expression (F) in whole splenocytes or purified T and B cells from WT and D18N mice (3 separations, 3 mice of each genotype). (G) IFN- expression in sorted naïve or differentiated CD44high CD4 or CD8 T cells from both WT and D18N mice (3 independent sorts, 3 animals of each genotype). All mice were mixed sex and 8–12 weeks of age. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001 by Log-rank test (A), unpaired student’s t-test (C, WT vs. D18N comparisons in D-G), or one-way ANOVA (B, D18N splenocytes vs. other D18N populations in D-G). Conclusions Our findings demonstrate that TREX1 enzymatic activity is crucial to prevent inappropriate DNA-sensing and IFN-I production. TREX1 inactivity within hematopoietic cells was both necessary and sufficient to induce lupus-like autoimmunity, indicating that TREX1 normally acts within immune cells to suppress inappropriate activation of anti-viral signaling. Both innate immune and T cells respond to TREX1 dysfunction by spontaneously synthesizing IFN-, a surprising result given that T cells are not canonically thought to be major IFN--producing cells. These results expand our understanding of the pathogenesis of lupus-like disease, and indicate that small molecule inhibition of TREX1 could represent an appealing strategy for anti-viral and cancer immune-therapies. Funding Source(s): NIH grant -- 5R01AI116725–03 NIH training grant -- 5T32AI007401–23

Background: Patients with chronic inflammatory conditions, such as autoimmune diseases, infections, and cancer, often display anemia of inflammation (AI). 50% of patients with systemic lupus erythematosus display clinical signs of anemia. Mutations at aspartate residue 18 of the three prime repair exonuclease 1 ( TREX1 ) gene, which codes for the primary mammalian exonuclease, cause a monogenic form of cutaneous lupus erythematosus in humans and the genetically precise TREX1 D18N mice recapitulate a lupus-like disease. Objective: The link between failed DNA degradation during erythropoiesis and anemia prompted our studies to investigate whether TREX1 dysfunction might contribute to AI. Methods: AI in TREX1 D18N mice was determined through analysis of hematocrit, hemoglobin, gene expression analysis, immunohistochemistry (IHC) and flow cytometry. Response to phenylhydrazine (PHZ) induced anemia and activation of DNA sensing pathways were determined through IHC and interferon stimulatory gene (ISG) expression analysis of the liver, spleen and erythroblastic islands. Results: The TREX1 D18N mice exhibit altered erythropoiesis including reduced hematocrit, increased erythropoietic and hematotoxic gene expression levels in the spleen (~10-20 fold) and phenotypic signs of normocytic normochromic anemia. Anemia in TREX1 lupus mice is accompanied by increased erythropoietin (Epo) and extramedullary hematopoiesis. Furthermore, the TREX1 D18N mice display an inappropriate response to anemic challenge. Enhanced ISG expression is apparent and results from failed processing and sensing of undegraded erythroblast DNA. Conclusion: These results support a critical role for TREX1 in the degradation of erythroblast DNA and identify a distinct AI phenotype in TREX1 D18N mice.

Anaemia is commonly observed in chronic inflammatory conditions, including systemic lupus erythematosus (SLE), where approximate to 50% of patients display clinical signs of anaemia. Mutation at the aspartate residue 18 of the three prime repair exonuclease 1 (TREX1) gene causes a monogenic form of cutaneous lupus in humans and the genetically precise TREX1 D18N mice recapitulate a lupus-like disease. TREX1 degrades single- and double-stranded DNA (dsDNA), and the link between failed DNA degradation by nucleases, including nucleoside-diphosphate kinases (NM23H1/H2) and Deoxyribonuclease II (DNase II), and anaemia prompted our studies to investigate whether TREX1 dysfunction contributes to anaemia. Utilizing the TREX1 D18N mice we demonstrate that (1) TREX1 mutant mice develop normocytic normochromic anaemia and (2) TREX1 exonuclease participates in the degradation of DNA originating from erythroblast nuclei during definitive erythropoiesis. Gene expression, hematocrit, hemoglobin, immunohistochemistry (IHC) and flow cytometry were used to quantify dysfunctional erythropoiesis. An altered response to induced anaemia in the TREX1 D18N mice was determined through IHC, flow cytometry, and interferon-stimulated gene (ISG) expression analysis of the liver, spleen and erythroblastic islands (EBIs). IHC, flow cytometry, and ISG expression studies were performed in vitro to determine the role of TREX1 in the degradation of erythroblast DNA within EBIs. The TREX1 D18N mice exhibit altered erythropoiesis including a 20% reduction in hematocrit, 10-20 fold increased erythropoietic gene expression levels in the spleen and phenotypic signs of normocytic normochromic anaemia. Anaemia in TREX1 D18N mice is accompanied by increased erythropoietin (Epo), normal hepcidin levels and the TREX1 D18N mice display an inappropriate response to anaemic challenge. Enhanced ISG expression results from failed processing and subsequent sensing of undegraded erythroblast DNA in EBIs. TREX1 participates in the degradation of erythroblast DNA in the EBI and TREX1 D18N mice exhibit a normocytic normochromic anaemia.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.

The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes amonogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme.

TREX1 is an autonomous 3-exonuclease that degrades DNA to prevent inappropriate immune activation. The TREX1 protein is composed of 314 amino acids; the N-terminal 242 amino acids contain the catalytic domain, and the C-terminal region (CTR) localizes TREX1 to the cytosolic compartment. In this study, we show that TREX1 modification by ubiquitination is controlled by a highly conserved sequence in the CTR to affect cellular localization. Transfection of TREX1 deletion constructs into human cells demonstrated that this sequence is required for ubiquitination at multiple lysine residues through a non-canonical ubiquitin linkage. A proteomic approach identified ubiquilin 1 as a TREX1 CTR-interacting protein, and this interaction was verified in vitro and in vivo. Cotransfection studies indicated that ubiquilin 1 localizes TREX1 to cytosolic punctate structures dependent upon the TREX1 CTR and lysines within the TREX1 catalytic core. Several TREX1 mutants linked to the autoimmune diseases Aicardi-Goutieres syndrome and systemic lupus erythematosus that exhibit full catalytic function were tested for altered ubiquitin modification and cellular localization. Our data show that these catalytically competent disease-causing TREX1 mutants exhibit differential levels of ubiquitination relative to WT TREX1, suggesting a novel mechanism of dysfunction. Furthermore, these differentially ubiquitinated disease-causing mutants also exhibit altered ubiquilin 1 co-localization. Thus, TREX1 post-translational modification indicates an additional mechanism by which mutations disrupt TREX1 biology, leading to human autoimmune disease.

Mutations within the human TREX1 3' exonuclease are associated with Aicardi-Goutières Syndrome (AGS) and familial chilblain lupus (FCL). Both AGS and FCL are autoimmune diseases that result in increased levels of interferon alpha and circulating antibodies to DNA. TREX1 is a member of the endoplasmic reticulum (ER)-associated SET complex and participates in granzyme A-mediated cell death to degrade nicked genomic DNA. The loss of TREX1 activity may result in the accumulation of double-stranded DNA (dsDNA) degradation intermediates that trigger autoimmune activation. The X-ray crystal structures of the TREX1 wt apoprotein, the dominant D200H, D200N and D18N homodimer mutants derived from AGS and FCL patients, as well as the recessive V201D homodimer mutant have been determined. The structures of the D200H and D200N mutant proteins reveal the enzyme has lost coordination of one of the active site metals, and the catalytic histidine (H195) is trapped in a conformation pointing away from the active site. The TREX1 D18N and V201D mutants are able to bind both metals in the active site, but with inter-metal distances that are larger than optimal for catalysis. Additionally, all of the mutant structures reveal a reduced mobility in the catalytic histidine, providing further explanation for the loss of catalytic activity. The structures of the mutant TREX1 proteins provide insight into the dysfunction relating to human disease. Additionally, the TREX1 apoprotein structure together with the previously determined wild type substrate and product structures allow us to propose a distinct mechanism for the TREX1 exonuclease.

Mutations in the TREX1 gene cause Aicardi-Goutieres syndrome (AGS) and are linked to the autoimmune disease systemic lupus erythematosus. The TREX1 protein is a dimeric 3' DNA exonuclease that degrades DNA to prevent inappropriate immune activation. One of the most common TREX1 mutations, R114H, causes AGS as a homozygous and compound heterozygous mutation and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1 proteins containing R114H and the insertion mutations aspartate at position 201 (D201ins) and alanine at position 124 (A124ins), found in compound heterozygous AGS with R114H, were prepared and the DNA degradation activities were tested. The homodimer TREX1(R114H/R114H) exhibits a 23-fold reduced single-stranded DNA (ssDNA) exonuclease activity relative to TREX1(WT). The TREX1(D201ins/D201ins) and TREX1(A124ins/A124ins) exhibit more than 10,000-fold reduced ssDNA degradation activities. However, the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers exhibit activities 10-fold greater than the TREX1(R114H/R114H) homodimer during ssDNA and double-stranded DNA (dsDNA) degradation. These higher levels of activities measured in the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers are attributed to Arg-114 residues of TREX1(D201ins) and TREX1(A124ins) positioned at the dimer interface contributing to the active sites of the opposing TREX1(R114H) protomer. This interpretation is further supported by exonuclease activities measured for TREX1 enzymes containing R114A and R114K mutations. These biochemical data provide direct evidence for TREX1 residues in one protomer contributing to DNA degradation catalyzed in the opposing protomer and help to explain the dimeric TREX1 structure required for full catalytic competency.

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.

Mutations in the genes encoding the RNaseH2 and TREX1 nucleases have been identified in patients with Aicardi-Goutieres syndrome (AGS). To determine if the AGS RNaseH2 mutations result in the loss of nuclease activity, the human wild-type RNaseH2 and four mutant complexes that constitute the majority of mutations identified in AGS patients have been prepared and tested for ribonuclease H activity. The heterotrimeric structures of the mutant RNaseH2 complexes are intact. Furthermore, the ribonuclease H activities of the mutant complexes are indistinguishable from the wild-type enzyme with the exception of the RNaseH2 subunit A (Gly37Ser) mutant, which exhibits some evidence of altered nuclease specificity. These data indicate that the mechanism of RNaseH2 dysfunction in AGS cannot be simply explained by loss of ribonuclease H activity and points to a more complex mechanism perhaps mediated through altered interactions with as yet identified nucleic acids or protein partners.

Mutations in TREX1 have been linked to a spectrum of human autoimmune diseases including Aicardi-Goutieres syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus, and retinal vasculopathy and cerebral leukodystrophy. A common feature in these conditions is the frequent detection of antibodies to double-stranded DNA (dsDNA). TREX1 participates in a cell death process implicating this major 3' -> 5' exonuclease in genomic DNA degradation to minimize potential immune activation by persistent self DNA. The TREX1 D200N and D18N dominant heterozygous mutations were identified in AGS and FCL, respectively. TREX1 enzymes containing the D200N and D18N mutations were compared using nicked dsDNA and single-stranded DNA (ssDNA) degradation assays. The TREX1(WT/D200N) and TREX1(WT/D18N) heterodimers are completely deficient at degrading dsDNA and degrade ssDNA at an expected similar to 2-fold lower rate than TREX1WT enzyme. Further, the D200N- and D18N-containing TREX1 homo-and heterodimers inhibit the dsDNA degradation activity of TREX1(WT) enzyme, providing a likely explanation for the dominant phenotype of these TREX1 mutant alleles in AGS and FCL. By comparison, the TREX1 R114H homozygous mutation causes AGS and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1(R114H/R114H) homodimer has dysfunctional dsDNA and ssDNA degradation activities and does not detectibly inhibit the TREX1(WT) enzyme, whereas the TREX1(WT/R114H) heterodimer has a functional dsDNA degradation activity, supporting the recessive genetics of TREX1 R114H in AGS. The dysfunctional dsDNA degradation activities of these disease-related TREX1 mutants could account for persistent dsDNA from dying cells leading to an aberrant immune response in these clinically related disorders.

The activity of human TREX2-catalyzed 3' --> 5'-deoxyribonuclease has been analyzed in steady-state and single turnover kinetic assays and in equilibrium DNA binding studies. These kinetic data provide evidence for cooperative DNA binding within TREX2 and for coordinated catalysis between the TREX2 active sites supporting a model for communication between the protomers of a TREX2 dimer. Mobile loops positioned adjacent to the active sites provide the major DNA binding contribution and facilitate subsequent binding into the active sites. Mutations of three arginine residues on these loops cause decreased TREX2 activities by up to 60-fold. Steady-state kinetic assays of these arginine to alanine TREX2 variants result in increased K(m) values for DNA substrate with no effect on k(cat) values indicating contributions exclusively to DNA binding by all three of the loop arginines. TREX2 heterodimers were prepared to determine whether exonuclease activity in one protomer is communicated to the opposing protomer. Evidence for communication across the dimer interface is provided by the 7-fold lower catalytic activity measured in the TREX2(WT/H188A) heterodimer compared with the TREX2(WT) homodimer, contrasting the 2-fold lower activity measured in the TREX2(WT/R163A,R165A,R167A) heterodimer. The measured activity in TREX2(WT/H188A) heterodimer indicates that defective catalysis in one protomer reduces activity in the opposing protomer. A DNA binding analysis of TREX2 and the heterodimers indicates a cooperative binding effect within the TREX2 protomer. Finally, single turnover kinetic assays identify DNA binding as the rate-limiting step in TREX2 catalysis.