B cell-derived cfDNA after primary BNT162b2 mRNA vaccination anticipates memory B cells and SARS-CoV-2 neutralizing antibodies.

BACKGROUND: Much remains unknown regarding the response of the immune system to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination. METHODS: We employed circulating cell-free DNA (cfDNA) to assess the turnover of specific immune cell types following administration of the Pfizer/BioNTech vaccine. FINDINGS: The levels of B cell cfDNA after the primary dose correlated with development of neutralizing antibodies and memory B cells after the booster, revealing a link between early B cell turnover-potentially reflecting affinity maturation-and later development of effective humoral response. We also observed co-elevation of B cell, T cell, and monocyte cfDNA after the booster, underscoring the involvement of innate immune cell turnover in the development of humoral and cellular adaptive immunity. Actual cell counts remained largely stable following vaccination, other than a previously demonstrated temporary reduction in neutrophil and lymphocyte counts. CONCLUSIONS: Immune cfDNA dynamics reveal the crucial role of the primary SARS-CoV-2 vaccine in shaping responses of the immune system following the booster vaccine. FUNDING: This work was supported by a generous gift from Shlomo Kramer. Supported by grants from Human Islet Research Network (HIRN UC4DK116274 and UC4DK104216 to R.S. and Y.D.), Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Alex U Soyka Pancreatic Cancer Fund, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation, the Helmsley Charitable Trust, Grail, and the DON Foundation (to Y.D.). Y.D. holds the Walter and Greta Stiel Chair and Research Grant in Heart Studies. I.F.-F. received a fellowship from the Glassman Hebrew University Diabetes Center.

Maternal Monocytes Respond to Cell-Free Fetal DNA and Initiate Key Processes of Human Parturition.

Throughout gestation, the maternal immune system is tightly modulated to allow growth of a semiallogeneic fetus. During the third trimester, the maternal immune system shifts to a proinflammatory phenotype in preparation for labor. What induces this shift remains unclear. Cell-free fetal DNA (cffDNA) is shed by the placenta and enters maternal circulation throughout pregnancy. Levels of cffDNA are increased as gestation progresses and peak before labor, coinciding with a shift to proinflammatory maternal immunity. Furthermore, cffDNA is abnormally elevated in plasma from women with complications of pregnancy, including preterm labor. Given the changes in maternal immunity at the end of pregnancy and the role of sterile inflammation in the pathophysiology of spontaneous preterm birth, we hypothesized that cffDNA can act as a damage-associated molecular pattern inducing an inflammatory cytokine response that promotes hallmarks of parturition. To test this hypothesis, we stimulated human maternal leukocytes with cffDNA from primary term cytotrophoblasts or maternal plasma and observed significant IL-1ß and CXCL10 secretion, which coincides with phosphorylation of IFN regulatory factor 3 and caspase-1 cleavage. We then show that human maternal monocytes are crucial for the immune response to cffDNA and can activate bystander T cells to secrete proinflammatory IFN-γ and granzyme B. Lastly, we find that the monocyte response to cffDNA leads to vascular endothelium activation, induction of myometrial contractility, and PGE2 release in vitro. Our results suggest that the immune response to cffDNA can promote key features of the parturition cascade, which has physiologic consequences relevant to the timing of labor.

Genomic and transcriptomic correlates of immunotherapy response within the tumor microenvironment of leptomeningeal metastases.

Leptomeningeal disease (LMD) is a devastating complication of solid tumor malignancies, with dire prognosis and no effective systemic treatment options. Over the past decade, the incidence of LMD has steadily increased due to therapeutics that have extended the survival of cancer patients, highlighting the need for new interventions. To examine the efficacy of immune checkpoint inhibitors (ICI) in patients with LMD, we completed two phase II clinical trials. Here, we investigate the cellular and molecular features underpinning observed patient trajectories in these trials by applying single-cell RNA and cell-free DNA profiling to longitudinal cerebrospinal fluid (CSF) draws from enrolled patients. We recover immune and malignant cell types in the CSF, characterize cell behavior changes following ICI, and identify genomic features associated with relevant clinical phenomena. Overall, our study describes the liquid LMD tumor microenvironment prior to and following ICI treatment and demonstrates clinical utility of cell-free and single-cell genomic measurements for LMD research.

Circulating Mitochondrial DNA Stimulates Innate Immune Signaling Pathways to Mediate Acute Kidney Injury.

Mitochondrial dysfunction is increasingly considered as a critical contributor to the occurrence and progression of acute kidney injury (AKI). However, the mechanisms by which damaged mitochondria mediate AKI progression are multifactorial and complicated. Mitochondrial DNA (mtDNA) released from damaged mitochondria could serve as a danger-associated molecular pattern (DAMP) and activate the innate immune system through STING, TLR9, NLRP3, and some other adaptors, and further mediate tubular cell inflammation and apoptosis. Accumulating evidence has demonstrated the important role of circulating mtDNA and its related pathways in the progression of AKI, and regulating the proteins involved in these pathways may be an effective strategy to reduce renal tubular injury and alleviate AKI. Here, we aim to provide a comprehensive overview of recent studies on mtDNA-mediated renal pathological events to provide new insights in the setting of AKI.

Autoantibody-mediated impairment of DNASE1L3 activity in sporadic systemic lupus erythematosus.

Antibodies to double-stranded DNA (dsDNA) are prevalent in systemic lupus erythematosus (SLE), particularly in patients with lupus nephritis, yet the nature and regulation of antigenic cell-free DNA (cfDNA) are poorly understood. Null mutations in the secreted DNase DNASE1L3 cause human monogenic SLE with anti-dsDNA autoreactivity. We report that >50% of sporadic SLE patients with nephritis manifested reduced DNASE1L3 activity in circulation, which was associated with neutralizing autoantibodies to DNASE1L3. These patients had normal total plasma cfDNA levels but showed accumulation of cfDNA in circulating microparticles. Microparticle-associated cfDNA contained a higher fraction of longer polynucleosomal cfDNA fragments, which bound autoantibodies with higher affinity than mononucleosomal fragments. Autoantibodies to DNASE1L3-sensitive antigens on microparticles were prevalent in SLE nephritis patients and correlated with the accumulation of cfDNA in microparticles and with disease severity. DNASE1L3-sensitive antigens included DNA-associated proteins such as HMGB1. Our results reveal autoantibody-mediated impairment of DNASE1L3 activity as a common nongenetic mechanism facilitating anti-dsDNA autoreactivity in patients with severe sporadic SLE.

Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions.

Consumption of Eurasian bovine meat and milk has been associated with cancer development, in particular with colorectal cancer (CRC). In addition, zoonotic infectious agents from bovine products were proposed to cause colon cancer (zur Hausen et al., 2009). Bovine meat and milk factors (BMMF) are small episomal DNA molecules frequently isolated from bovine sera and milk products, and recently, also from colon cancer (de Villiers et al., 2019). BMMF are bioactive in human cells and were proposed to induce chronic inflammation in precancerous tissue leading to increased radical formation: for example, reactive oxygen and reactive nitrogen species and elevated levels of DNA mutations in replicating cells, such as cancer progenitor cells (zur Hausen et al., 2018). Mouse monoclonal antibodies against the replication (Rep) protein of H1MSB.1 (BMMF1) were used to analyze BMMF presence in different cohorts of CRC peritumor and tumor tissues and cancer-free individuals by immunohistochemistry and Western blot. BMMF DNA was isolated by laser microdissection from immunohistochemistry-positive tissue regions. We found BMMF Rep protein present specifically in close vicinity of CD68+ macrophages in the interstitial lamina propria adjacent to CRC tissues, suggesting the presence of local chronic inflammation. BMMF1 (modified H1MSB.1) DNA was isolated from the same tissue regions. Rep and CD68+ detection increased significantly in peritumor cancer tissues when compared to tissues of cancer-free individuals. This strengthens previous postulations that BMMF function as indirect carcinogens by inducing chronic inflammation and DNA damage in replicating cells, which represent progress to progenitor cells for adenoma (polyps) formation and cancer.

Development of a Specific Monoclonal Antibody to Detect Male Cells Expressing the RPS4Y1 Protein.

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.

Donor-derived cell-free DNA (dd-cfDNA) for detection of allograft rejection in pediatric kidney transplants.

In pediatric transplantation, acute rejection is a major contributor of graft failure. Current approaches include kidney biopsy in response to graft dysfunction and/or the emergence of donor-specific HLA antibodies (DSA). However, biopsy is associated with potential complications. Thus, there is a need for non-invasive diagnostics. Detection of donor-derived cell-free DNA (dd-cfDNA, AlloSure) > 1% is associated with rejection in adult kidney transplants. Here, we evaluate the utility of dd-cfDNA for identifying allograft rejection in pediatric patients. Between 10/2017 and 10/2019, 67 patients, who underwent initial testing with dd-cfDNA as part of routine monitoring or in response to clinical suspicion for rejection, were included. Biopsies were performed when dd-cfDNA > 1.0% or where clinical suspicion was high. Demographics, dd-cfDNA, antibody status, and biopsies were collected prospectively. Data were analyzed to determine predictive value of dd-cfDNA for identifying grafts at risk for rejection. 19 of 67 patients had dd-cfDNA testing as part of routine monitoring with a median dd-cfDNA score of 0.37 (IQR: 0.19-1.10). 48 of 67 patients who had clinical suspicion of rejection had median dd-cfDNA score of 0.47 (0.24-2.15). DSA-positive recipients had higher dd-cfDNA scores than those who were negative or had AT1R positivity alone (P = .003). There was no association between dd-cfDNA score and strength of DSA positivity. 7 of 48 recipients had a biopsy with a dd-cfDNA score <1%; two showed evidence of rejection. Neither DSA nor AT1R positivity was statistically associated with biopsy-proven rejection. However, dd-cfDNA >1% was diagnostic of rejection with sensitivity of 86% and specificity of 100% (AUC: 0.996, 0.98-1.00; P = .002). dd-cfDNA represents a non-invasive method for early detection of rejection in pediatric renal transplants. Our study shows dd-cfDNA to be highly predictive of histological rejection and superior to other indicators such as graft dysfunction or antibody positivity alone. Further studies are necessary to refine these initial observations.

Oxidized cell-free DNA as a stress-signaling factor activating the chronic inflammatory process in patients with autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASD) are known to be associated with an inflammatory process related to immune system dysfunction. This study's aim was to investigate the role of cell-free DNA in chronic inflammatory process in ASD patients. METHODS: The study included 133 ASD patients and 27 healthy controls. Sixty-two ASD patients were demonstrated to have mild-to-moderate disease severity (group I) and 71 individuals to have severe ASD (group II). Plasma cell-free (cf) DNA characteristics, plasma cytokine concentrations, expression of the genes for NFкB1 transcription factor and pro-inflammatory cytokines TNFα, IL-1ß and IL-8 in peripheral blood lymphocytes (PBL) of ASD patients, and unaffected controls were investigated. Additionally, in vitro experiments with oxidized DNA supplementation to PBL cultures derived from ASD patients and healthy controls were performed. RESULTS: The data indicates that ASD patients have demonstrated increased cfDNA concentration in their circulation. cfDNA of patients with severe ASD has been characterized by a high abundance of oxidative modification. Furthermore, ASD patients of both groups have shown elevated plasma cytokine (IL-1ß, IL-8, IL-17A) levels and heightened expression of genes for NFкB1 nuclear factor and pro-inflammatory cytokines TNFα, IL-1ß, and IL-8 in PBL. In vitro experiments have shown that NF-κB/cytokine mRNA expression profiles of ASD patient PBL treated with oxidized DNA fragments were significantly different from those of healthy controls. CONCLUSIONS: It may be proposed that oxidized cfDNA plays a role of stress-signaling factor activating the chronic inflammatory process in patients with ASD.

Adding Insult on Injury: Immunogenic Role for Donor-derived Cell-free DNA?

Donor-derived cell-free DNA (dd-cfDNA) in the blood circulation is an early marker of injury in solid organ transplantation. Here, we review recent evidence that indicates that dd-cfDNA may itself be a trigger of inflammation, thereby adding insult on injury. Early unresolving molecular allograft injury measured via changes in dd-cfDNA may be an early warning sign and may therefore enable stratification of patients who are at risk of subsequent allograft injury. Considering dd-cfDNA as a continuous and clinically significant biomarker opens up the potential for new management strategies, therapeutics, and ways to quantify interventions by considering the immunological potential of dd-cfDNA.

Resolution of Deep Venous Thrombosis: Proposed Immune Paradigms.

Venous thromboembolism (VTE) is a pathology encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE) associated with high morbidity and mortality. Because patients often present after a thrombus has already formed, the mechanisms that drive DVT resolution are being investigated in search of treatment. Herein, we review the current literature, including the molecular mechanisms of fibrinolysis and collagenolysis, as well as the critical cellular roles of macrophages, neutrophils, and endothelial cells. We propose two general models for the operation of the immune system in the context of venous thrombosis. In early thrombus resolution, neutrophil influx stabilizes the tissue through NETosis. Meanwhile, macrophages and intact neutrophils recognize the extracellular DNA by the TLR9 receptor and induce fibrosis, a complimentary stabilization method. At later stages of resolution, pro-inflammatory macrophages police the thrombus for pathogens, a role supported by both T-cells and mast cells. Once they verify sterility, these macrophages transform into their pro-resolving phenotype. Endothelial cells both coat the stabilized thrombus, a necessary early step, and can undergo an endothelial-mesenchymal transition, which impedes DVT resolution. Several of these interactions hold promise for future therapy.

The Role of Extracellular DNA and Histones in Ischaemia-Reperfusion Injury of the Myocardium.

Despite an increase in the rates of survival in patients suffering myocardial infarction, as yet there is no therapy specifically targeting ischaemia and reperfusion injury of the myocardium. With a greater understanding of immune activation during infarction, more potential treatment targets are now being identified. The innate immune system is believed to play an important role in the myocardium after ischaemia-driven cardiomyocyte death. The release of intracellular contents including DNA into the extracellular space during necrosis and cell rupture is now believed to create a pro-inflammatory milieu which propagates the inflammatory process. DNA and DNA fragments have been shown to activate the innate immune system by acting as Danger-Associated Molecular Patterns (DAMPs), which act as ligands on toll-like receptors (TLRs). Stimulation of TLRs, in turn, can activate intracellular cell death pathways such as pyroptosis. Here, we review the role of DNA fragments during ischaemia and reperfusion, and assess their potential as a target in the quest to preserve cardiomyocyte viability following myocardial infarction.

Monoclonal anti-dsDNA antibody 2C10 escorts DNA to intracellular DNA sensors in normal mononuclear cells and stimulates secretion of multiple cytokines implicated in lupus pathogenesis.

There have been many studies on the mechanisms of internalization of DNA-anti-DNA immune complexes by cells, including the one used for rheumatoid factor-expressing mouse B cells. In parallel, studies on the role of intracellular DNA sensors in the pathogenesis of systemic lupus erythematosus (SLE) have been conducted, including the one using a mouse model lacking one of the sensors. These and other data have established a framework for understanding the pathogenic role of anti-DNA antibodies, but studies on normal cells are limited. Here, we used the monoclonal anti-dsDNA antibody 2C10, 2-kbp dsDNA and healthy human peripheral blood mononuclear cells (PBMCs) to test whether and how 2C10 and/or DNA cause pathology in normal cells. We found that on culture with PBMCs, 2C10 preferentially entered monocytes and that DNA enhanced this internalization. In contrast, DNA alone was not significantly internalized by monocytes, but 2C10 facilitated its internalization. This was suppressed by cytochalasin D, but not by methyl-ß-cyclodextrin, chloroquine or an Fc blocker, suggesting the involvement of macropinocytosis in this process. Internalization of 2C10 and DNA together resulted in production of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1ß, IL-6, IL-10 and IL-33 by PBMCs. Cytokine production was suppressed by chloroquine and shikonin, but not by RU.521, suggesting dependence on activation of the Toll-like receptor (TLR)-9 and absent in melanoma 2 (AIM-2) pathways. These results established a simple model to demonstrate that anti-DNA antibodies can cause dysregulation of cytokine network mimicking systemic lupus erythematosus in culture of normal PBMCs, and emphasize again the importance of maintaining anti-DNA antibodies at low levels by treatment.

Systemic Lupus Erythematosus and DNA Degradation and Elimination Defects.

Introduction: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by the production of autoantibodies specific for components of the cell nucleus and that causes damage to body tissues and organs. The pathogenesis of SLE remains unclear, with numerous studies pointing to a combination of genetic and environmental factors. A critical stage in SLE development is cell necrosis, in which undegraded chromatin and nucleoproteins are released into the blood, resulting in circulating cell-free DNA and serum nucleoproteins that trigger anti-dsDNA autoantibody production. This systematic literature review aimed to examine whether SLE stems from a DNA degradation and elimination defect. Materials and Methods: An advanced literature search was conducted in PubMed using the following keywords: [("SLE" OR "Systemic Lupus Erythematosus" OR "Lupus")] AND [("DNA" OR "DNA Degradation")] AND [("Defect Elimination")]. More articles were obtained from the references of the identified articles and basic Google searches. Twenty-five peer-reviewed articles published within the past 10 years (2007-2018) were included for review. Results: The findings of each study are summarized in Tables 1, 2. Discussion and Conclusion: The etiopathogenesis of SLE remains controversial, which limits therapeutic inventions for this disease. However, SLE is a DNA degradation and elimination disorder caused by uncleared histones and nuclear material that leak into the extracellular space and form cell-free DNA, triggering an immune response that destroys tissues and organs. Under normal conditions, apoptosis allows DNA and other nuclear material to be efficiently cleared through degradation and additional complex mechanisms such that this material does not trigger the immune system to produce nuclear autoantibodies.

Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients - A pilot study.

The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.

Toll-Like Receptor 9 Plays a Pivotal Role in Angiotensin II-Induced Atherosclerosis.

Background Toll-like receptor ( TLR ) 9 recognizes bacterial DNA , activating innate immunity, whereas it also provokes inflammation in response to fragmented DNA released from mammalian cells. We investigated whether TLR 9 contributes to the development of vascular inflammation and atherogenesis using apolipoprotein E-deficient ( Apoe -/-) mice. Methods and Results Tlr9-deficient Apoe -/- ( Tlr9 -/- Apoe -/-) mice and Apoe -/- mice on a Western-type diet received subcutaneous angiotensin II infusion (1000 ng/kg per minute) for 28 days. Angiotensin II increased the plasma level of double-stranded DNA, an endogenous ligand of TLR 9, in these mice. Genetic deletion or pharmacologic blockade of TLR 9 in angiotensin II-infused Apoe -/- mice attenuated atherogenesis in the aortic arch ( P<0.05), reduced the accumulation of lipid and macrophages in atherosclerotic plaques, and decreased RNA expression of inflammatory molecules in the aorta with no alteration of metabolic parameters. On the other hand, restoration of TLR 9 in bone marrow in Tlr9 -/- Apoe -/- mice promoted atherogenesis in the aortic arch ( P<0.05). A TLR 9 agonist markedly promoted proinflammatory activation of Apoe -/- macrophages, partially through p38 mitogen-activated protein kinase signaling. In addition, genomic DNA extracted from macrophages promoted inflammatory molecule expression more effectively in Apoe -/- macrophages than in Tlr9 -/- Apoe -/- macrophages. Furthermore, in humans, circulating double-stranded DNA in the coronary artery positively correlated with inflammatory features of coronary plaques determined by optical coherence tomography in patients with acute myocardial infarction ( P<0.05). Conclusions TLR 9 plays a pivotal role in the development of vascular inflammation and atherogenesis through proinflammatory activation of macrophages. TLR 9 may serve as a potential therapeutic target for atherosclerosis.

New insight into the role of extracellular vesicles in kidney disease.

Extracellular vesicles (EVs) are released to maintain cellular homeostasis as well as to mediate cell communication by spreading protective or injury signals to neighbour or remote cells. In kidney, increasing evidence support that EVs are signalling vesicles for different segments of tubules, intra-glomerular, glomerular-tubule and tubule-interstitial communication. EVs released by kidney resident and infiltrating cells can be isolated from urine and were found to be promising biomarkers for kidney disease, reflecting deterioration of renal function and histological change. We have here summarized the recent progress about the functional role of EVs in kidney disease as well as challenges and future directions involved.

Changes in the biochemical taste of cytoplasmic and cell-free DNA are major fuels for inflamm-aging.

Inflamm-aging depicts the progressive activation of the innate immune system that accompanies human aging. Its role as a disease-predisposing condition has been proposed, but its molecular basis is still poorly understood. A wealth of literature conveys that, particularly upon stress, nuclear and mitochondrial genomes are released into the cytoplasmic and extracellular compartments. Cytoplasmic (cy) and cell-free (cf) DNA pools trigger inflammation and innate immunity at local and systemic level. In particular, cyDNA plays a crucial role in the phenomenon of cell senescence and in the cognate pro-inflammatory secretome. Here we propose that changes in a variety of biochemical characteristics "tastes" of cy- and cf-DNA (e.g. the amount of 8-oxo-deoxy-guanosine and 5-methyl-deoxy-cytosine, the proportion of DNA hybridized with RNA) potentially affect the capability of these DNA pools to ignite the innate immune system. We also underpin that telomeric sequences are major components of the cy/cfDNA payload. Telomere shortening, a hallmark of aging, causes the depletion of telomeric sequences in cy/cfDNA pool, thus unleashing their potential to exert an age-related activation of the innate immune system. Finally, we posit that various sources of DNA (extracellular vesicles, the commensal metagenome and food) contribute to the cy/cfDNA payloads. We speculate that changes in the biochemical "taste" of cy/cfDNA are major modifiers of inflamm-aging.

Systemic activation of NLRP3 inflammasome in patients with severe primary Sjögren's syndrome fueled by inflammagenic DNA accumulations.

Sjögren's syndrome (SS) patients manifest high cell-free DNA (cf-DNA) levels in serum, associated with impaired DNaseI activity. Undegraded DNA may accumulate in tissues and act as an inflammasome-activating signal. Herein, we investigated the occurrence of aberrant DNA build-up in various biologic compartments of SS patients and its correlation with the activity of NLRP3 and AIM2 inflammasomes. For this purpose, we evaluated sera, PBMC, circulating monocytes and salivary glands (SG) from different SS patient subgroups and controls. We found that SS patients at high risk for lymphoma and those with established lymphoma display high serum cf-DNA levels, substantial extranuclear DNA accumulations in PBMC and SG tissues, a unique NLRP3 inflammasome gene signature in PBMC, and significantly increased serum IL-18 and ASC levels. In these patients, the circulating monocytes manifested NLRP3 inflammasome activation and increased response to NLRP3 stimuli, whereas SG-infiltrating macrophages exhibited signs of NLRP3 activation and pyroptosis. Cell-free nucleic acids isolated from patients' sera competently primed the activation of both NLRP3 and AIM2 inflammasomes in healthy monocytes. SS patients also manifested diminished DNaseI activity in serum and DNaseII expression in PBMC, which inversely correlated with indices of inflammasome activation. DNaseII gene-silencing in healthy monocytes led to cytoplasmic DNA deposition and activation of inflammasome-related genes and of caspase1. Our data reveal the occurrence of systemic NLRP3 inflammasome activation in severe SS, which is associated with widespread extranuclear accumulations of inflammagenic DNA and impaired DNA degradation. These findings can provide novel biomarkers and new therapeutic targets for the management of SS patients with adverse outcomes.