NETosis, Coagulation, and Neutrophil Dysfunction
1 Krinsky et al. 2023 — NETosis Induction Reflects COVID-19 Severity and Long COVID
Full Citation:: Krinsky N, Levy S, Zisman-Ilani Y, Mandelboim M, Nili A, et al. NETosis induction reflects COVID-19 severity and long COVID: insights from a 2-center patient cohort study in Israel. Journal of Thrombosis and Haemostasis. 2023;21(9):2561–2575. (Krinsky et al. 2023) DOI:: 10.1016/j.jtha.2023.02.033 PMID:: 37054916 Study Design:: Two-centre prospective cohort Sample Size:: \(n=177\) patients (acute mild/moderate, acute severe/critical, convalescent recovered, convalescent long COVID) \(+\) 54 non-COVID controls Key Findings::
- NETosis induction capacity correlated with COVID-19 severity and persisted in long COVID patients months after acute resolution
- NETosis induction was more sensitive than MPO-DNA levels for distinguishing disease severity strata
- Elevated NETosis correlated with platelet activation markers and coagulation factor levels
- Dexamethasone treatment reduced NETosis induction, suggesting corticosteroids partially act via NET suppressionRelevance:: Provides clinical cohort evidence that NETosis is a sustained pathogenic driver in Long COVID (and by extension post-viral ME/CFS), not merely a transient acute-phase response. Establishes NETosis induction capacity as a candidate biomarker for ongoing post-viral pathology. Certainty Assessment::
- *Quality:* High (Journal of Thrombosis and Haemostasis, two-centre cohort)
- *Sample:* $n=177$ patients $+$ 54 controls
- *Replication:* Partially replicated (consistent with Romano2022 and Monsalve2025)
- *Certainty:* 0.752 Monsalve et al. 2025 — NETosis: A Key Player in Autoimmunity, COVID-19, and Long COVID
Full Citation:: Monsalve DM, Acosta-Ampudia Y, Guerrero Acosta N, Celis-Andrada M, ahin A, Morva Yilmaz A, Shoenfeld Y, Ram'irez-Santana C. NETosis: A key player in autoimmunity, COVID-19, and long COVID. Journal of Translational Autoimmunity. 2025;10:100280. (Monsalve et al. 2025) DOI:: 10.1016/j.jtauto.2025.100280 PMID:: 40071133 Study Design:: Systematic review Key Findings::
- NET components (citrullinated histones, extracellular DNA) act as persistent autoantigens driving autoimmune-like pathology
- SARS-CoV-2 spike protein directly triggers NETosis via ACE2 on neutrophils
- NETs activate NLRP3 inflammasome, sustaining IL-1$\beta$/IL-18 release in Long COVID
- Therapeutic targets identified: PAD4 inhibitors (reduce citrullination), recombinant DNase I (enhance clearance), ACPA blockadeRelevance:: Most comprehensive current review of the NETosis-autoimmunity bridge. Provides mechanistic grounding for why ME/CFS patients who develop new post-viral autoantibodies may have NET-generated citrullinated proteins as the upstream antigenic trigger. Certainty Assessment::
- *Quality:* High (Journal of Translational Autoimmunity, systematic review)
- *Replication:* Synthesises findings across multiple independent groups
- *Certainty:* 0.70Full Citation:: Sch"onrich G, Raftery MJ. Neutrophil Extracellular Traps Go Viral. Frontiers in Immunology. 2016;7:366. (Schönrich and Raftery 2016) DOI:: 10.3389/fimmu.2016.00366 PMID:: 27698656 Study Design:: Narrative review Key Findings::
- Viruses trigger NETosis via both direct PAMP recognition and indirect cytokine signaling (IFN, TNF)
- NETs immobilize virions and amplify innate immune recruitment
- Herpesviruses (EBV, CMV, HSV) are established viral NETosis inducers
- Some viruses suppress NETosis as an immune evasion mechanism
- Excessive viral-triggered NETosis causes bystander tissue damage beyond infection controlRelevance:: Foundational mechanistic reference establishing that viruses implicated in ME/CFS triggering (particularly EBV and CMV) are direct NETosis inducers. Provides the upstream link in the viral-trigger → NETosis → chronic inflammation chain. Certainty Assessment::
- *Quality:* Medium (Frontiers in Immunology, 2016 narrative review; well-cited)
- *Certainty:* 0.55 (background/foundational reference)3 Hetland et al. 2022 — NETs and Calprotectin Levels Correlate with Severity After ChAdOx1 Vaccination
Full Citation:: Hetland G, Fagerhol MK, Wiedmann MKH, et al. Elevated NETs and Calprotectin Levels after ChAdOx1 nCoV-19 Vaccination Correlate with the Severity of Side Effects. Vaccines. 2022;10(8):1267. (Hetland et al. 2022) DOI:: 10.3390/vaccines10081267 PMID:: 36016155 Study Design:: Case-comparison study Key Findings::
- H3-NET levels and calprotectin (S100A8/S100A9) were markedly elevated in VITT patients vs.\ mild post-vaccination symptoms
- Strong correlation between NET/calprotectin levels and clinical severity ($r \geq 0.745$, $p < 0.001$)
- Establishes calprotectin as a clinically accessible proxy for NET-mediated thromboinflammationRelevance:: Demonstrates that NETosis-driven thromboinflammation operates across diverse immune stimuli. Calprotectin measurement is a practical, inexpensive potential biomarker for NET burden that could be applied to ME/CFS monitoring. Certainty Assessment::
- *Quality:* Medium (Vaccines journal, small VITT cohort)
- *Sample:* Small VITT case series
- *Certainty:* 0.504 Weckbach et al. 2019 — Midkine Drives Cardiac Inflammation via NETosis in Myocarditis
Full Citation:: Weckbach LT, Grabmaier U, Uhl A, et al. Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis. The Journal of Experimental Medicine. 2019;216(5):1040–1058. (Weckbach et al. 2019) DOI:: 10.1084/jem.20181102 PMID:: 30647120 Study Design:: Human tissue analysis \(+\) mouse model (experimental myocarditis) Key Findings::
- NETs identified in cardiac tissue from human myocarditis patients and mouse models
- Midkine–LRP1 axis specifically recruits neutrophils to inflamed cardiac tissue and drives NETosis
- NET formation promotes fibrosis and ventricular dysfunction
- Blocking midkine or LRP1 reduces cardiac NET deposition and injuryRelevance to ch10:: Provides mechanism for how NETs cause tissue-level cardiac injury in post-viral conditions. Relevant to post-COVID/ME/CFS subclinical cardiomyopathy; midkine and LRP1 are potential therapeutic targets. Certainty Assessment::
- *Quality:* High (Journal of Experimental Medicine; human tissue + validated mouse model)
- *Certainty:* 0.65
- *Limitation:* Translational relevance to ME/CFS cardiac involvement is inferential5 Veras et al. 2023 — Targeting NETs Reduces Multiple Organ Injury in a COVID-19 Mouse Model
Full Citation:: Veras FP, Gomes GF, Silva BMS, Caeté DB, Almeida CJLR, Silva CMS, Schneider AH, Corneo ES, Bonilha CS, Batah SS, Martins R, Arruda E, Fabro AT, Alves-Filho JC, Cunha TM, Cunha FQ. Targeting neutrophils extracellular traps (NETs) reduces multiple organ injury in a COVID-19 mouse model. Respiratory Research. 2023;24(1):66. (Veras et al. 2023) DOI:: 10.1186/s12931-023-02336-2 PMID:: 36864506 Study Design:: Mouse model (SARS-CoV-2 infection) \(+\) in vitro lung epithelial cell assay Key Findings::
- DNase I treatment reduced detectable NET levels and improved clinical disease in SARS-CoV-2-infected mice
- Purified NETs directly damaged lung epithelial cells in vitro; DNase I mitigated this
- NET disruption protected lungs, heart, and kidneys simultaneously — suggesting NETs mediate multi-organ injuryRelevance:: Provides in vivo proof-of-concept that pharmacological NET clearance (via DNase I) reduces organ injury downstream of viral infection. Supports DNase I or related NET-clearing agents as candidate therapeutics for post-viral multi-system conditions including ME/CFS. Certainty Assessment::
- *Quality:* Medium-High (Respiratory Research; rigorous mouse model with mechanism confirmed in vitro)
- *Certainty:* 0.55 (mouse model; human translation pending)
- *Limitation:* Animal model results require clinical translation6 Garcia et al. 2024 — Impaired NET/DNase Balance in COVID-19 Disease
Full Citation:: Garcia G, Labrouche-Colomer S, Duvignaud A, Clequin E, Dussiau C, Trégouët DA, Malvy D, Prevel R, Zouine A, Pellegrin I, Goret J, Mamani-Matsuda M, Dewitte A, James C. Impaired balance between neutrophil extracellular trap formation and degradation by DNases in COVID-19 disease. Journal of Translational Medicine. 2024;22:246. (Garcia et al. 2024) DOI:: 10.1186/s12967-024-05044-7 PMID:: 38454482 Study Design:: Cross-sectional cohort (French multicentre) Sample Size:: \(n=145\) total (93 ambulatory, 15 severe, 37 critical) + 21 healthy donors Key Findings::
- Functional DNase levels were diminished in severe and critical COVID-19 patients compared to ambulatory cases
- NET markers (MPO-DNA, H3cit, H3cit-DNA complexes) correlated positively with CRP and neutrophil/lymphocyte ratio
- NET/DNase ratios were markedly elevated in severe patients, indicating insufficient NET clearance
- DNase1 antigen was lower in severe/critical vs.\ ambulatory COVID-19; DNase1L3 antigen did not rise proportionally
- Nine linked *DNASE1* polymorphisms associated with ~75% reduction in DNase1 antigen (all carriers were critical patients)
- Plasmacytoid dendritic cell (pDC) counts reduced in critical patients; scRNAseq showed reduced *DNASE1L3* expression in pDCs with increasing severity
- H3cit and H3cit-DNA levels predicted hospitalisation among ambulatory patientsRelevance:: First demonstration that NET/DNase imbalance drives disease severity in COVID-19 via insufficient DNase upregulation, pDC depletion, and genetic susceptibility. Establishes DNase1/DNase1L3 deficiency and pDC dysfunction as mechanisms that could contribute to persistent NET-mediated thrombo-inflammation in post-viral ME/CFS. Certainty Assessment::
- *Quality:* High (Journal of Translational Medicine, multicentre cohort with mechanistic genetic and flow cytometric validation)
- *Sample:* $n=145$ patients + 21 controls
- *Replication:* Partially replicated (lower DNase activity confirmed in independent cohorts)
- *Certainty:* 0.70
- *Limitation:* Cross-sectional; NET/DNase dynamics in ME/CFS not directly measured7 Sanhueza et al. 2026 — De Novo Insulin Resistance Drives NETosis in Long COVID
Full Citation:: Sanhueza S, Cabrera C, Quiroga R, et al. De novo COVID-19-associated insulin resistance drives dysregulated neutrophil extracellular trap formation (NETosis) four months after infection. Frontiers in Immunology. 2026;17:1787799. (Sanhueza et al. 2026) DOI:: 10.3389/fimmu.2026.1787799 PMID:: 42158877 Study Design:: Prospective cohort + in vitro mechanistic assays Sample Size:: \(n=60\) COVID-19 patients stratified by metabolic status at 4-month follow-up Key Findings::
- 24 of 36 patients without pre-existing glucose disorders developed *de novo* insulin resistance (IR) at 4 months post-COVID
- IR patients exhibited increased basal NETosis but paradoxically impaired NETosis response to TLR7/8 agonists
- Plasma from IR patients significantly enhanced NETosis in control neutrophils in vitro
- Insulin enhanced NETosis independently of glucose concentrations in vitro
- Glycolysis-driven neutrophil metabolism is particularly sensitive to systemic metabolic disturbancesRelevance:: Establishes post-infectious insulin resistance as a metabolic driver of sustained NETosis. This links metabolic dysfunction (glycolysis impairment, insulin resistance) to NET-mediated thrombo-inflammation, connecting the ME/CFS energy metabolism and immune dysregulation domains via a testable pathway. Certainty Assessment::
- *Quality:* Medium-High (Frontiers in Immunology; prospective design with in vitro mechanistic validation)
- *Sample:* $n=60$ (modest for subgroup analyses)
- *Replication:* Not yet independently replicated
- *Certainty:* 0.60
- *Limitation:* Single-centre (Chile); no DNase measurements; metabolic-NET axis in ME/CFS not tested8 Thierry et al. 2025 — NETs Structurally Associated with Microclots in Long COVID
Full Citation:: Thierry AR, Usher T, Sanchez C, et al. Circulating microclots are structurally associated with neutrophil extracellular traps and their amounts are elevated in long COVID patients. Journal of Medical Virology. 2025;97(10):e70613. (Thierry et al. 2025) DOI:: 10.1002/jmv.70613 PMID:: 41036702 Study Design:: Cross-sectional case-control Key Findings::
- NET markers (myeloperoxidase, neutrophil elastase, circulating DNA) quantitatively and structurally associated with circulating microclots
- Strong diagnostic performance for discriminating Long COVID from healthy controls (both individually and combined)
- NETs appear to be a structural component of microclots, potentially promoting microclot stabilisation
- NET-microclot association provides mechanistic bridge between thrombo-inflammation and microvascular obstructionRelevance:: Demonstrates that NETs are physically incorporated into microclots, providing a structural basis for persistent microvascular pathology in post-viral conditions. Relevant to ME/CFS microclot hypotheses and orthostatic intolerance (cerebral hypoperfusion) mechanisms. Certainty Assessment::
- *Quality:* Medium (Journal of Medical Virology; novel structural findings)
- *Replication:* Partially replicated (microclot and NET findings converge across multiple Long COVID studies)
- *Certainty:* 0.65
- *Limitation:* Cross-sectional; DNase-specific measurements not reported; sample size unclear from abstract9 Perez Mazzali et al. 2026 — Persistent T Cell and Innate Immune Dysregulation in Long COVID
Full Citation:: Perez Mazzali M, Pérez-Cózar F, Cal-Sabater P, et al. Persistent T cell phenotypic alterations and early innate immune dysregulation as potential biomarkers of long COVID. The Journal of Infection. 2026;92(5):106731. (Perez Mazzali et al. 2026) DOI:: 10.1016/j.jinf.2026.106731 PMID:: 41856437 Study Design:: Prospective longitudinal case-control (acute phase + 3-month follow-up) Key Findings::
- Distinct immune signatures discriminated Long COVID from non-Long COVID patients
- Early abnormalities in innate immunity during acute infection predicted Long COVID development
- Dendritic cell abnormalities observed (subset-specific dysfunction in antigen-presenting cells)
- Persistent alterations in adaptive immune cell subsets at 3 months post-discharge
- Mass cytometry (40-marker panel) enabled high-dimensional immune profilingRelevance:: Establishes that early innate immune dysregulation (including dendritic cell abnormalities) predicts post-acute outcomes. Relevant to ME/CFS biomarker research and pDC/DNase deficiency hypotheses — if dendritic cell dysfunction is an early predictor, DNase1L3 deficiency may be an early rather than late feature. Certainty Assessment::
- *Quality:* Medium (Journal of Infection; prospective design, high-dimensional profiling)
- *Certainty:* 0.55
- *Limitation:* pDC-specific data not detailed; no NET or DNase measurements; abstract-level assessment10 Augustin et al. 2026 — Gut-Immune Axis Dysregulation in Post-COVID Syndrome
Full Citation:: Augustin M, Picard L, Rauschning D, et al. Persistent gut-immune axis dysregulation in long-term post-COVID syndrome: Insights from a prospective, observational, cross-sectional case-control study. Mucosal Immunology. 2026. (Augustin et al. 2026) DOI:: 10.1016/j.mucimm.2026.03.002 PMID:: 41794369 Study Design:: Cross-sectional case-control with terminal ileum biopsies Sample Size:: \(n=43\) convalescent patients (20 PCS+, 23 PCS−) Key Findings::
- Expansion of plasmacytoid dendritic cells (pDCs) in terminal ileum of PCS+ patients
- Elevated SARS-CoV-2 nucleocapsid protein expression in terminal ileum tissue
- Increased mast cell activity and zonulin levels (gut barrier dysfunction)
- Alterations in NK cell subsets and central memory T cells
- Elevated MMP-9 levels indicating localised inflammation and tissue remodellingRelevance:: Complicates the pDC/DNase1L3 hypothesis: Garcia 2024 showed pDC depletion in acute COVID-19 blood, but Augustin 2026 shows pDC expansion in Long COVID gut tissue. Possible temporal dynamics (depletion → expansion), tissue compartment differences (blood vs. gut), or functional impairment despite numerical expansion. Relevant to ME/CFS gut-immune axis hypotheses. Certainty Assessment::
- *Quality:* Medium (Mucosal Immunology; novel tissue-based approach)
- *Sample:* $n=43$ total ($n=20$ PCS+)
- *Replication:* pDC findings contradictory to Garcia 2024 (temporal/compartment difference likely)
- *Certainty:* 0.55
- *Limitation:* Small sample; no NET/DNase measurements; invasive biopsy required