Elien Van Wonterghem

Passenger Mutations Confound Interpretation of All Genetically Modified Congenic Mice

Targeted mutagenesis in mice is a powerful tool for functional analysis of genes. However, genetic variation between embryonic stem cells (ESCs) used for targeting (previously almost exclusively 129-derived) and recipient strains (often C57BL/6J) typically results in congenic mice in which the targeted gene is flanked by ESC-derived passenger DNA potentially containing mutations. Comparative genomic analysis of 129 and C57BL/6J mouse strains revealed indels and single nucleotide polymorphisms resulting in alternative or aberrant amino acid sequences in 1,084 genes in the 129-strain genome. Annotating these passenger mutations to the reported genetically modified congenic mice that were generated using 129-strain ESCs revealed that nearly all these mice possess multiple passenger mutations potentially influencing the phenotypic outcome. We illustrated this phenotypic interference of 129-derived passenger mutations with several case studies and developed a Me-PaMuFind-It web tool to estimate the number and possible effect of passenger mutations in transgenic mice of interest.

Matrix Metalloprotease 8-Dependent Extracellular Matrix Cleavage at the Blood–CSF Barrier Contributes to Lethality during Systemic Inflammatory Diseases

Systemic inflammatory response syndrome (SIRS) is a highly mortal inflammatory disease, associated with systemic inflammation and organ dysfunction. SIRS can have a sterile cause or can be initiated by an infection, called sepsis. The prevalence is high, and available treatments are ineffective and mainly supportive. Consequently, there is an urgent need for new treatments. The brain is one of the first organs affected during SIRS, and sepsis and the consequent neurological complications, such as encephalopathy, are correlated with decreased survival. The choroid plexus (CP) that forms the blood–CSF barrier (BCSFB) is thought to act as a brain “immune sensor” involved in the communication between the peripheral immune system and the CNS. Nevertheless, the involvement of BCSFB integrity in systemic inflammatory diseases is seldom investigated. We report that matrix metalloprotease-8 (MMP8) depletion or inhibition protects mice from death and hypothermia in sepsis and renal ischemia/reperfusion. This effect could be attributed to MMP8-dependent leakage of the BCSFB, caused by collagen cleavage in the extracellular matrix of CP cells, which leads to a dramatic change in cellular morphology. Disruption of the BCSFB results in increased CSF cytokine levels, brain inflammation, and downregulation of the brain glucocorticoid receptor. This receptor is necessary for dampening the inflammatory response. Consequently, MMP8+/+ mice, in contrast to MMP8−/− mice, show no anti-inflammatory response and this results in high mortality. In conclusion, we identify MMP8 as an essential mediator in SIRS and, hence, a potential drug target. We also propose that the mechanism of action of MMP8 involves disruption of the BCSFB integrity.

Simultaneous Targeting of IL-1 and IL-18 Is Required for Protection against Inflammatory and Septic Shock

RATIONALE Sepsis is one of the leading causes of death around the world. The failure of clinical trials to treat sepsis demonstrates that the molecular mechanisms are multiple and are still insufficiently understood. OBJECTIVES To clarify the long disputed hierarchical contribution of several central inflammatory mediators (IL-1β, IL-18, caspase [CASP] 7, CASP1, and CASP11) in septic shock and to explore their therapeutic potential. METHODS LPS- and tumor necrosis factor (TNF)-induced lethal shock, and cecal ligation and puncture (CLP) were performed in genetically or pharmacologically targeted mice. Body temperature and survival were monitored closely, and plasma was analyzed for several markers of cellular disintegration and inflammation. MEASUREMENTS AND MAIN RESULTS Interestingly, deficiency of both IL-1β and IL-18 additively prevented LPS-induced mortality. The detrimental role of IL-1β and IL-18 was confirmed in mice subjected to a lethal dose of TNF, or to a lethal CLP procedure. Although their upstream activator, CASP1, and its amplifier, CASP11, are considered potential therapeutic targets because of their crucial involvement in endotoxin-induced toxicity, CASP11- or CASP1/11-deficient mice were not, or hardly, protected against a lethal TNF or CLP challenge. In line with our results obtained in genetically deficient mice, only the combined neutralization of IL-1 and IL-18, using the IL-1 receptor antagonist anakinra and anti-IL-18 antibodies, conferred complete protection against endotoxin-induced lethality. CONCLUSIONS Our data point toward the therapeutic potential of neutralizing IL-1 and IL-18 simultaneously in sepsis, rather than inhibiting the upstream inflammatory caspases.

Matrix metalloproteinase 13 modulates intestinal epithelial barrier integrity in inflammatory diseases by activating TNF.

Several pathological processes, such as sepsis and inflammatory bowel disease (IBD), are associated with impairment of intestinal epithelial barrier. Here, we investigated the role of matrix metalloproteinase MMP13 in these diseases. We observed that MMP13−/− mice display a strong protection in LPS‐ and caecal ligation and puncture‐induced sepsis. We could attribute this protection to reduced LPS‐induced goblet cell depletion, endoplasmic reticulum stress, permeability and tight junction destabilization in the gut of MMP13−/− mice compared to MMP13+/+ mice. Both in vitro and in vivo, we found that MMP13 is able to cleave pro‐TNF into bioactive TNF. By LC‐MS/MS, we identified three MMP13 cleavage sites, which proves that MMP13 is an alternative TNF sheddase next to the TNF converting enzyme TACE. Similarly, we found that the same mechanism was responsible for the observed protection of the MMP13−/− mice in a mouse model of DSS‐induced colitis. We identified MMP13 as an important mediator in sepsis and IBD via the shedding of TNF. Hence, we propose MMP13 as a novel drug target for diseases in which damage to the gut is essential.

Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus-derived extracellular vesicles.

Here, we identified release of extracellular vesicles (EVs) by the choroid plexus epithelium (CPE) as a new mechanism of blood–brain communication. Systemic inflammation induced an increase in EVs and associated pro‐inflammatory miRNAs, including miR‐146a and miR‐155, in the CSF. Interestingly, this was associated with an increase in amount of multivesicular bodies (MVBs) and exosomes per MVB in the CPE cells. Additionally, we could mimic this using LPS‐stimulated primary CPE cells and choroid plexus explants. These choroid plexus‐derived EVs can enter the brain parenchyma and are taken up by astrocytes and microglia, inducing miRNA target repression and inflammatory gene up‐regulation. Interestingly, this could be blocked in vivo by intracerebroventricular (icv) injection of an inhibitor of exosome production. Our data show that CPE cells sense and transmit information about the peripheral inflammatory status to the central nervous system (CNS) via the release of EVs into the CSF, which transfer this pro‐inflammatory message to recipient brain cells. Additionally, we revealed that blockage of EV secretion decreases brain inflammation, which opens up new avenues to treat systemic inflammatory diseases such as sepsis.

Amyloid β Oligomers Disrupt Blood–CSF Barrier Integrity by Activating Matrix Metalloproteinases

The blood–CSF barrier (BCSFB) consists of a monolayer of choroid plexus epithelial (CPE) cells that maintain CNS homeostasis by producing CSF and restricting the passage of undesirable molecules and pathogens into the brain. Alzheimers disease is the most common progressive neurodegenerative disorder and is characterized by the presence of amyloid β (Aβ) plaques and neurofibrillary tangles in the brain. Recent research shows that Alzheimers disease is associated with morphological changes in CPE cells and compromised production of CSF. Here, we studied the direct effects of Aβ on the functionality of the BCSFB. Intracerebroventricular injection of Aβ1–42 oligomers into the cerebral ventricles of mice, a validated Alzheimers disease model, caused induction of a cascade of detrimental events, including increased inflammatory gene expression in CPE cells and increased levels of proinflammatory cytokines and chemokines in the CSF. It also rapidly affected CPE cell morphology and tight junction protein levels. These changes were associated with loss of BCSFB integrity, as shown by an increase in BCSFB leakage. Aβ1–42 oligomers also increased matrix metalloproteinase (MMP) gene expression in the CPE and its activity in CSF. Interestingly, BCSFB disruption induced by Aβ1–42 oligomers did not occur in the presence of a broad-spectrum MMP inhibitor or in MMP3-deficient mice. These data provide evidence that MMPs are essential for the BCSFB leakage induced by Aβ1–42 oligomers. Our results reveal that Alzheimers disease-associated soluble Aβ1–42 oligomers induce BCSFB dysfunction and suggest MMPs as a possible therapeutic target. SIGNIFICANCE STATEMENT No treatments are yet available to cure Alzheimers disease; however, soluble Aβ oligomers are believed to play a crucial role in the neuroinflammation that is observed in this disease. Here, we studied the effect of Aβ oligomers on the often neglected barrier between blood and brain, called the blood–CSF barrier (BCSFB). This BCSFB is formed by the choroid plexus epithelial cells and is important in maintaining brain homeostasis. We observed Aβ oligomer-induced changes in morphology and loss of BCSFB integrity that might play a role in Alzheimers disease progression. Strikingly, both inhibition of matrix metalloproteinase (MMP) activity and MMP3 deficiency could protect against the detrimental effects of Aβ oligomer. Clearly, our results suggest that MMP inhibition might have therapeutic potential.

Pharmacological Inhibition of Type I Interferon Signaling Protects Mice against Lethal Sepsis

Current research on new therapeutic strategies for sepsis uses different animal models, such as the lipopolysaccharide-induced endotoxemia model and the cecal ligation and puncture (CLP) peritonitis model. By using genetic and pharmacologic inhibition of the type I interferon (IFN) receptor (IFNAR1), we show that type I IFN signaling plays a detrimental role in these sepsis models. Mortality after CLP was reduced even when type I IFN responses were blocked after the onset of sepsis. Our findings reveal that type I IFNs play an important detrimental role during sepsis by negatively regulating neutrophil recruitment. Reduced neutrophil influx likely occurs via the induction of the CXC motif chemokine 1. Moreover, human white blood cells exposed to heat-killed Pseudomonas aeruginosa secrete IFN-β and stimulate type I IFN signaling. We provide data that support pharmacologic inhibition of type I IFN signaling as a novel therapeutic treatment in severe sepsis.

Comparing exosome-like vesicles with liposomes for the functional cellular delivery of small RNAs

Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natural carriers for biomolecule transfer between cells. This unique feature rationalizes their exploitation as bio-inspired drug delivery systems. However, the therapeutic application of ELVs is hampered by the lack of efficient and reproducible drug loading methods, in particular for therapeutic macromolecules. To overcome this limitation, we present a generic method to attach siRNA to the surface of isolated ELVs by means of a cholesterol anchor. Despite a feasible uptake in both a dendritic and lung epithelial cell line, B16F10- and JAWSII-derived ELVs were unable to functionally deliver the associated small RNAs, neither exogenous cholesterol-conjugated siRNA nor endogenous miRNA derived from the melanoma producer cell. The latter results were confirmed both for purified ELVs and ELVs delivered via a transwell co-culture set-up. In contrast, simple anionic fusogenic liposomes were able to induce a marked siRNA-mediated gene knockdown under equal experimental conditions, both indicating successful cytosolic delivery of surface-bound cholesterol-conjugated siRNA and further underscoring the incapacity of the here evaluated ELVs to guide cytosolic delivery of small RNAs. In conclusion, we demonstrate that a more in-depth understanding of the biomolecular delivery mechanism and specificity is required before ELVs can be envisioned as a generic siRNA carrier.

Counteracting the effects of TNF receptor‐1 has therapeutic potential in Alzheimer's disease

Alzheimers disease (AD) is the most common form of dementia, and neuroinflammation is an important hallmark of the pathogenesis. Tumor necrosis factor (TNF) might be detrimental in AD, though the results coming from clinical trials on anti‐TNF inhibitors are inconclusive. TNFR1, one of the TNF signaling receptors, contributes to the pathogenesis of AD by mediating neuronal cell death. The blood–cerebrospinal fluid (CSF) barrier consists of a monolayer of choroid plexus epithelial (CPE) cells, and AD is associated with changes in CPE cell morphology. Here, we report that TNF is the main inflammatory upstream mediator in choroid plexus tissue in AD patients. This was confirmed in two murine AD models: transgenic APP/PS1 mice and intracerebroventricular (icv) AβO injection. TNFR1 contributes to the morphological damage of CPE cells in AD, and TNFR1 abrogation reduces brain inflammation and prevents blood–CSF barrier impairment. In APP/PS1 transgenic mice, TNFR1 deficiency ameliorated amyloidosis. Ultimately, genetic and pharmacological blockage of TNFR1 rescued from the induced cognitive impairments. Our data indicate that TNFR1 is a promising therapeutic target for AD treatment.

Antisense oligonucleotides against TNFR1 prevent toxicity of TNF/IFNγ treatment in mouse tumor models

Tumor necrosis factor (TNF) has remarkable antitumor effects, but its systemic therapeutic use is prevented by its lethal inflammatory effects. TNFR1 (P55) is essential for both the antitumor and toxic effects because both of them are absent in P55‐deficient mice. In previous work we demonstrated that P55+/− mice are completely resistant to TNF toxicity, while the antitumor effects induced by TNF combined with interferon gamma (IFNγ) remain fully functional in these mice. Hence, a high dose of TNF/IFNγ has an excellent therapeutic potential when P55 levels are reduced, because TNF induces tumor regression without systemic toxicity. Here, we provide proof of principle for therapeutic application of this approach by using antisense oligonucleotides (ASOs). Treatment of mice with ASOs targeting P55 resulted in a strong reduction in P55 protein levels in liver, small intestine and blood mononuclear cells. This P55 downregulation was associated with significant protection of mice against acute TNF toxicity as measured by hypothermia, systemic inflammation and lethality. This treatment also protected mice against toxicity of TNF/IFNγ treatment in several cancer models: B16Bl6, Lewis lung carcinoma and a lung colony model. Our results confirm the therapeutic value of this strategy, which could lead to the development of a safer and more effective TNF/IFNγ antitumor therapy.