Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Cellular Imaging and Time-Domain FLIM Studies of Meso-Tetraphenylporphine Disulfonate as a Photosensitising Agent in 2D and 3D Models.

Fluorescence lifetime imaging (FLIM) and confocal fluorescence studies of a porphyrin-based photosensitiser (meso-tetraphenylporphine disulfonate: TPPS2a) were evaluated in 2D monolayer cultures and 3D compressed collagen constructs of a human ovarian cancer cell line (HEY). TPPS2a is known to be an effective model photosensitiser for both Photodynamic Therapy (PDT) and Photochemical Internalisation (PCI). This microspectrofluorimetric study aimed firstly to investigate the uptake and subcellular localisation of TPPS2a, and evaluate the photo-oxidative mechanism using reactive oxygen species (ROS) and lipid peroxidation probes combined with appropriate ROS scavengers. Light-induced intracellular redistribution of TPPS2a was observed, consistent with rupture of endolysosomes where the porphyrin localises. Using the same range of light doses, time-lapse confocal imaging permitted observation of PDT-induced generation of ROS in both 2D and 3D cancer models using fluorescence-based ROS together with specific ROS inhibitors. In addition, the use of red light excitation of the photosensitiser to minimise auto-oxidation of the probes was investigated. In the second part of the study, the photophysical properties of TPPS2a in cells were studied using a time-domain FLIM system with time-correlated single photon counting detection. Owing to the high sensitivity and spatial resolution of this system, we acquired FLIM images that enabled the fluorescence lifetime determination of the porphyrin within the endolysosomal vesicles. Changes in the lifetime dynamics upon prolonged illumination were revealed as the vesicles degraded within the cells.

Activation and Contraction of Human "Vascular" Smooth Muscle Cells Grown From Circulating Blood Progenitors.

Blood outgrowth smooth muscle cells (BO-SMCs) offer the means to study vascular cells without the requirement for surgery providing opportunities for drug discovery, tissue engineering, and personalized medicine. However, little is known about these cells which meant that their therapeutic potential remains unexplored. Our objective was to investigate for the first time the ability of BO-SMCs and vessel-derived smooth muscle cells to sense the thromboxane mimetic U46619 by measuring intracellular calcium elevation and contraction. U46619 (10-6 M) increased cytosolic calcium in BO-SMCs and vascular smooth muscle cells (VSMCs) but not in fibroblasts. Increased calcium signal peaked between 10 and 20 s after U46619 in both smooth muscle cell types. Importantly, U46619 (10-9 to 10-6 M) induced concentration-dependent contractions of both BO-SMCs and VSMCs but not in fibroblasts. In summary, we show that functional responses of BO-SMCs are in line with VSMCs providing critical evidence of their application in biomedical research.

Strong CD4 T Cell Responses to Zika Virus Antigens in a Cohort of Dengue Virus Immune Mothers of Congenital Zika Virus Syndrome Infants.

Background: There is an urgent need to understand the complex relationship between cross-reactive anti-viral immunity, disease susceptibility, and severity in the face of differential exposure to related, circulating Flaviviruses. Co-exposure to Dengue virus and Zika virus in Brazil is a case in point. A devastating aspect of the 2015-2016 South American Zika outbreak was the dramatic increase in numbers of infants born with microcephaly to mothers exposed to Zika virus during pregnancy. It has been proposed that this is more likely to ensue from Zika infection in women lacking cross-protective Dengue immunity. In this case series we measure the prevalence of Dengue immunity in a cohort of mothers exposed to Zika virus during pregnancy in the 2015-2016 Zika outbreak that gave birth to an infant affected by microcephaly and explore their adaptive immunity to Zika virus. Results: Fifty women from Sergipe, Brazil who gave birth to infants with microcephaly following Zika virus exposure during the 2015-16 outbreak were tested for serological evidence of Dengue exposure and IFNγ ELISpot spot forming cell (SFC) response to Zika virus. The majority (46/50) demonstrated Dengue immunity. IFNγ ELISpot responses to Zika virus antigens showed the following hierarchy: Env>NS1>NS3>C protein. Twenty T cell epitopes from Zika virus Env were identified. Responses to Zika virus antigens Env and NS1 were polyfunctional with cells making IFNγ, TNFα, IL-4, IL-13, and IL-10. In contrast, responses to NS5 only produced the immune regulatory TGFß1 cytokine. There were SFC responses against Zika virus Env (1-20) and variant peptide sequences from West Nile virus, Dengue virus 1-4 and Yellow Fever virus. Conclusion: Almost all the women in our study showed serological evidence of Dengue immunity, suggesting that microcephaly can occur in DENV immune mothers. T cell immunity to Zika virus showed a multifunctional response to the antigens Env and NS1 and immune regulatory responses to NS5 and C protein. Our data support an argument that different viral products may skew the antiviral response to a more pro or anti-inflammatory outcome, with an associated impact on immunopathogenesis.