Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Identifying a core protein signature of small extracellular vesicles derived from B-cell precursor acute lymphoblastic leukaemia.

Acute paediatric leukaemia is diagnosed and monitored via bone marrow aspirate assessment of blasts as a measure of minimal residual disease. Liquid biopsies in the form of blood samples could greatly reduce the need for invasive bone marrow aspirations, but there are currently no blood markers that match the sensitivity of bone marrow diagnostics. Circulating extracellular vesicles (EVs) represent candidate biomarkers that may reflect the blast burden in bone marrow, and several studies have reported on the utility of EVs as biomarkers for adult haematological malignancies. Increased levels of EVs have been reported for several haematological malignancies, and we similarly report here elevated EV concentrations in plasma from paediatric BCP-ALL patients. Plasma EVs are very heterogeneous in terms of their cellular origin, thus identifying a cancer selective EV-marker is challenging. Here, we undertook a reductionistic approach to identify protein markers selectively associated to plasma EVs derived from BCP-ALL patients. The EV proteome of primary BCP-ALL cell-derived EVs were compared against EVs from healthy donor B cells and the BCP-ALL cell line REH, and further against EVs isolated from plasma of healthy paediatric donors and paediatric BCP-ALL patients. With this approach, we identified a signature of 6 proteins (CD317, CD38, IGF2BP1, PCNA, CSDE1, and GPR116) that were specifically identified in BCP-ALL derived EVs only and not in healthy control EVs, and that could be exploited as diagnostic biomarkers.

Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins.

NK cells can broadly target and kill malignant cells via release of cytolytic proteins. NK cells also release extracellular vesicles (EVs) that contain cytolytic proteins, previously shown to induce apoptosis of a variety of cancer cells in vitro and in vivo. The EVs released by NK cells are likely very heterogeneous, as vesicles can be released from the plasma membrane or from different intracellular compartments. In this study, we undertook a fractionation scheme to enrich for cytolytic NK-EVs. NK-EVs were harvested from culture medium from the human NK-92 cell line or primary human NK cells grown in serum-free conditions. By combining ultracentrifugation with downstream density-gradient ultracentrifugation or size-exclusion chromatography, distinct EV populations were identified. Density-gradient ultracentrifugation led to separation of three subpopulations of EVs. The different EV isolates were characterized by label-free quantitative mass spectrometry and western blotting, and we found that one subpopulation was primarily enriched for plasma membrane proteins and tetraspanins CD37, CD82, and CD151, and likely represents microvesicles. The other major subpopulation was enriched in intracellularly derived markers with high expression of the endosomal tetraspanin CD63 and markers for intracellular organelles. The intracellularly derived EVs were highly enriched in cytolytic proteins, and possessed high apoptotic activity against HCT-116 colon cancer spheroids. To further enrich for cytolytic EVs, immunoaffinity pulldowns led to the isolation of a subset of EVs containing the cytolytic granule marker NKG7 and the majority of vesicular granzyme B content. We therefore propose that EVs containing cytolytic proteins may primarily be released via cytolytic granules.

Inflammasomes and SARS-CoV-2 Infection.

SARS-CoV-2 is a new type of coronavirus that has caused worldwide pandemic. The disease induced by SARS-CoV-2 is called COVID-19. A majority of people with COVID-19 have relatively mild respiratory symptoms. However, a small percentage of COVID-19 patients develop a severe disease where multiple organs are affected. These severe forms of SARS-CoV-2 infections are associated with excessive production of pro-inflammatory cytokines, so called "cytokine storm". Inflammasomes, which are protein complexes of the innate immune system orchestrate development of local and systemic inflammation during virus infection. Recent data suggest involvement of inflammasomes in severe COVID-19. Activation of inflammasome exerts two major effects: it activates caspase-1-mediated processing and secretion of pro-inflammatory cytokines IL-1ß and IL-18, and induces inflammatory cell death, pyroptosis, via protein called gasdermin D. Here, we provide comprehensive review of current understanding of the activation and possible functions of different inflammasome structures during SARS-CoV-2 infection and compare that to response caused by influenza A virus. We also discuss how novel SARS-CoV-2 mRNA vaccines activate innate immune response, which is a prerequisite for the activation of protective adaptive immune response.

Proteomic study of apheresis platelets made HLA class I deficient for transfusion of refractory patients.

PURPOSE: Refractoriness can occur after repeated platelet (PLT) transfusions because of alloimmunization to HLA class I antigens on transfused PLTs and generation of anti-HLA antibodies that bind to the foreign PLTs and initiate their destruction. Such refractoriness can be overcome by provision of HLA-matched PLTs from HLA typed donors. However, since the procedure is both expensive and time-consuming, an alternative approach is to deplete PLTs of HLA class I molecules by a brief treatment with citric acid, on ice. This is shown to be feasible without damaging PLT function. We used label free quantitative mass spectrometry (MS)-based proteomics to investigate the effect of acid treatment on apheresis PLTs for combatting immunologic PLT refractoriness. EXPERIMENTAL DESIGN: Proteomic analyses are undertaken using PLTs from seven apheresis concentrates, which were split in two with or without acid treatment. RESULTS: In total 1717 proteins in apheresis PLTs were quantified using proteomics. Data are available via ProteomeXchange with identifier PXD027893 . Of these, the amount of 80 proteins changed significantly after acid treatment, but overall there were not any major differences in proteomes between samples with and without acid treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In general, the changes of PLT proteins after treatment with citric acid were quite small and functionally safe. Hence, this result taken together with our previously published data indicates that acid treated PLTs can be used for treatment of patients with PLT refractoriness and opens up for a clinical trial.

Low Cellular NAD+ Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes.

NAD+ is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD+ on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD+ affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD+ was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD+ levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD+ enhanced IL-1ß release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD+ depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD+ depletion, specifically TAK1, IKKß, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD+ affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD+ and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.