14 research outputs found
Exploring new applications for photophysically encoded microcarriers
The first goal of this thesis was to investigate the potential of two cationic polysaccharides (PQ-4 and PQ-10) for DNA delivery. We have shown that, compared to PEI based polyplexes, they were less efficient in transfecting cells. However, as they had very low toxicity, further tailoring of the nature and extent of cationic side chains on cationic hydroxyethylcellulose may be a promising avenue to further enhance their DNA delivery properties. As a second goal we investigated the applications of digitally encoded microcarriers for cell based assays. We succeeded to show that encoded microcarriers were suitable to grow cells on. Neither the coating at the surface of the beads (which facilitates the growth of the cells), nor the cells themselves hampered the decoding of the beads, even when the cells covering the microcarriers exhibited green or red fluorescence due to the expression of GFP and RFP respectively. We were able (a) to immobilize DNA, siRNA or adenoviral particles on the surfaces of the encoded microcarriers by the use of polyelectrolytes and, subsequently, (b) to grow cells on top of the nucleic acids/adenoviral particles. The DNA and siRNA immobilized on the surface of the microcarrier were not able to transfect cells. However, we showed that the cells growing on the polyelectrolyte layer could indeed become transduced with adenoviral particles hosted by the polyelectrolyte layer. In conclusion, a proof of principal to use photophysically encoded microcarriers as transfected microarray has been shown. As a third goal we investigated the use of digitally encoded microcarriers as tool to combat counterfeiting of tablets. We showed that the codes in the Memobeads in tables produced by granulation did not deform during tabletting and that the code in the beads remained readable. We also found evidence that, after oral intake, the encoded microparticles are highly unlikely toxic to humans
The ubiquitin-editing enzyme A20 controls NK cell homeostasis through regulation of mTOR activity and TNF
The ubiquitin-editing enzyme A20 is a well-known regulator of immune cell function and homeostasis. In addition, A20 protects cells from death in an ill-defined manner. While most studies focus on its role in the TNF-receptor complex, we here identify a novel component in the A20-mediated decision between life and death. Loss of A20 in NK cells led to spontaneous NK cell death and severe NK cell lymphopenia. The few remaining NK cells showed an immature, hyperactivated phenotype, hallmarked by the basal release of cytokines and cytotoxic molecules. NK-A20(-/-) cells were hypersensitive to TNF-induced cell death and could be rescued, at least partially, by a combined deficiency with TNF. Unexpectedly, rapamycin, a well-established inhibitor of mTOR, also strongly protected NK-A20(-/-) cells from death, and further studies revealed that A20 restricts mTOR activation in NK cells. This study therefore maps A20 as a crucial regulator of mTOR signaling and underscores the need for a tightly balanced mTOR pathway in NK cell homeostasis
The ORMDL3 asthma susceptibility gene regulates systemic ceramide levels without altering key asthma features in mice
Background: Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)-like protein isoform 3 (ORMDL3) gene across different populations. Although the ORM homologues in yeast are well-known inhibitors of sphingolipid synthesis, it is still unclear whether and how mammalian ORMDL3 regulates sphingolipid metabolism and whether altered sphingolipid synthesis would be causally related to asthma risk.
Objective: We sought to examine the in vivo role of ORMDL3 in sphingolipid metabolism and allergic asthma.
Methods: Ormdl3-LacZ reporter mice, gene-deficient Ormdl3(-/-) mice, and overexpressing Ormdl3(Tg/wt) mice were exposed to physiologically relevant aeroallergens, such as house dust mite (HDM) or Alternaria alternata, to induce experimental asthma. Mass spectrometry-based sphingolipidomics were performed, and airway eosinophilia, T(H)2 cytokine production, immunoglobulin synthesis, airway remodeling, and bronchial hyperreactivity were measured.
Results: HDM challenge significantly increased levels of total sphingolipids in the lungs of HDM-sensitized mice compared with those in control mice. In Ormdl3(Tg/wt) mice the allergen-induced increase in lung ceramide levels was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, levels of total sphingolipids, including ceramides, were increased in Ormdl3(-/-) mice, whereas they were decreased in Ormdl3(Tg/wt) mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wildtype, Ormdl3(Tg/wt), and Ormdl3(-/-) mice across several models of experimental asthma.
Conclusion: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma
STE20 kinase TAOK3 regulates type 2 immunity and metabolism in obesity
Healthy adipose tissue (AT) contains ST2(+) Tregs, ILC2s, and alternatively activated macrophages that are lost in mice or humans on high caloric diet. Understanding how this form of type 2 immunity is regulated could improve treatment of obesity. The STE20 kinase Thousand And One amino acid Kinase-3 (TAOK3) has been linked to obesity in mice and humans, but its precise function is unknown. We found that ST2(+) Tregs are upregulated in visceral epididymal white AT (eWAT) of Taok3(-/-) mice, dependent on IL-33 and the kinase activity of TAOK3. Upon high fat diet feeding, metabolic dysfunction was attenuated in Taok3(-/-) mice. ST2(+) Tregs disappeared from eWAT in obese wild-type mice, but this was not the case in Taok3(-/-) mice. Mechanistically, AT Taok3(-/-) Tregs were intrinsically more responsive to IL-33, through higher expression of ST2, and expressed more PPAR & gamma; and type 2 cytokines. Thus, TAOK3 inhibits adipose tissue Tregs and regulates immunometabolism under excessive caloric intake.
Maes et al. reveal an unexpected role of TAOK3 in regulating ST2(+) regulatory T cells in mouse adipose tissue. Absence of TAOK3 sustains Tregs in obesity and improves metabolic dysfunction
Influence of plasmid DNA topology on the transfection properties of DOTAP/DOPE lipoplexes
Physicochemical and transfection properties of cationic hydroxyethylcellulose/DNA nanoparticles
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Canonical IRE1 function needed to sustain vigorous natural killer cell proliferation during viral infection
The unfolded protein response (UPR) aims to restore ER homeostasis under conditions of high protein folding load, a function primarily serving secretory cells. Additional, non-canonical UPR functions have recently been unraveled in immune cells. We addressed the function of the inositol-requiring enzyme 1 (IRE1) signaling branch of the UPR in NK cells in homeostasis and microbial challenge. Cell-intrinsic compound deficiency of IRE1 and its downstream transcription factor XBP1 in NKp46+ NK cells, did not affect basal NK cell homeostasis, or overall outcome of viral MCMV infection. However, mixed bone marrow chimeras revealed a competitive advantage in the proliferation of IRE1-sufficient Ly49H+ NK cells after viral infection. CITE-Seq analysis confirmed strong induction of IRE1 early upon infection, concomitant with the activation of a canonical UPR signature. Therefore, we conclude that IRE1/XBP1 activation is required during vigorous NK cell proliferation early upon viral infection, as part of a canonical UPR response.</p