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Resistance of Collagenase-2 (Matrix Metalloproteinase-8)-Deficient Mice to TNF-Induced Lethal Hepatitis

Acute fulminant liver failure is a serious worldwide health problem. Despite maximal supportive intensive care treatment, the disease offers a poor prognosis, with mortality rates of >80%. We have previously shown that a broad-spectrum inhibitor of matrix metalloproteinases (MMPs) confers complete protection in a mouse model of TNF-induced lethal hepatitis, thereby suggesting the possibility of protecting cancer patients against the deleterious side effects of TNF therapy. In our search for the individual matrix metalloproteinases involved, we found that the recently generated MMP-8-deficient mice are significantly protected against TNF-induced acute hepatitis. In contrast to their wild-type counterparts, MMP-8-null mice display very little hepatocyte necrosis and apoptosis, resulting in a much better survival outcome. We found that these animals clearly display impaired leukocyte influx into the liver and no release of the neutrophil-specific, LPS-induced CXC chemokine. Our findings provide evidence that MMP-8 plays an essential role in acute liver failure and might be a promising new target for the treatment for this illness.

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.

Safe TNF-based antitumor therapy following p55TNFR reduction in intestinal epithelium

TNF has remarkable antitumor activities; however, therapeutic applications have not been possible because of the systemic and lethal proinflammatory effects induced by TNF. Both the antitumor and inflammatory effects of TNF are mediated by the TNF receptor p55 (p55TNFR) (encoded by the Tnfrsf1a gene). The antitumor effect stems from an induction of cell death in tumor endothelium, but the cell type that initiates the lethal inflammatory cascade has been unclear. Using conditional Tnfrsf1a knockout or reactivation mice, we found that the expression level of p55TNFR in intestinal epithelial cells (IECs) is a crucial determinant in TNF-induced lethal inflammation. Remarkably, tumor endothelium and IECs exhibited differential sensitivities to TNF when p55TNFR levels were reduced. Tumor-bearing Tnfrsf1a⁺⁺/⁻ or IEC-specific p55TNFR-deficient mice showed resistance to TNF-induced lethality, while the tumor endothelium remained fully responsive to TNF-induced apoptosis and tumors regressed. We demonstrate proof of principle for clinical application of this approach using neutralizing anti-human p55TNFR antibodies in human TNFRSF1A knockin mice. Our results uncover an important cellular basis of TNF toxicity and reveal that IEC-specific or systemic reduction of p55TNFR mitigates TNF toxicity without loss of antitumor efficacy.

Generation and Characterization of Small Single Domain Antibodies Inhibiting Human Tumor Necrosis Factor Receptor 1

Background: Several anti-TNF biologicals are available to treat autoimmune diseases. However, selective TNFR1 inhibition is advisable, thereby reducing the pro-inflammatory TNF/TNFR1 signaling, while the good immunomodulatory TNF/TNFR2 signaling is preserved. Results: We generated and characterized an anti-TNFR1 Nanobody, TNF Receptor-One Silencer (TROS). Conclusion: TROS inhibits inflammation in vitro, ex vivo, and in vivo. Significance: Anti-TNFR1 therapies are potential novel treatments against autoimmune diseases. The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named “TNF Receptor-One Silencer” (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

LPS resistance of SPRET/Ei mice is mediated by Gilz, encoded by the Tsc22d3 gene on the X chromosome

Natural variation for LPS‐induced lethal inflammation in mice is useful for identifying new genes that regulate sepsis, which could form the basis for novel therapies for systemic inflammation in humans. Here we report that LPS resistance of the inbred mouse strain SPRET/Ei, previously reported to depend on the glucocorticoid receptor (GR), maps to the distal region of the X‐chromosome. The GR‐inducible gene Tsc22d3, encoding the protein Gilz and located in the critical region on the X‐chromosome, showed a higher expressed SPRET/Ei allele, regulated in cis. Higher Gilz levels were causally related to reduced inflammation, as shown with knockdown and overexpression studies in macrophages. Transient overexpression of Gilz by hydrodynamic plasmid injection confirmed that Gilz protects mice against endotoxemia Our data strongly suggest that Gilz is responsible for the LPS resistance of SPRET/Ei mice and that it could become a treatment option for sepsis.

Hyporesponsiveness of SPRET/Ei mice to lethal shock induced by tumor necrosis factor and implications for a TNF-based antitumor therapy.

Tumor necrosis factor (TNF) is a central mediator in lethal shock and an interesting cytokine for anticancer therapy. To inhibit TNF-induced lethal shock, it is important to identify protective genes. Here we demonstrate that the SPRET/Ei mouse strain, derived from Mus spretus, exhibits an extremely dominant resistance to TNF-induced lethal inflammation. An interspecific backcross experiment revealed that the TNF hyporesponse is linked to loci on chromosomes 2, 6, and 11. Treatment of inoculated tumors with TNF and IFN-γ leads to regression and a highly reduced toxicity in (C57BL/6 × SPRET/Ei)F1 mice.

Regulation and dysregulation of tumor necrosis factor receptor-1

TNF is an essential regulator of the immune system. Dysregulation of TNF plays a role in the pathology of many auto-immune diseases. TNF-blocking agents have proven successful in the treatment of such diseases. Development of novel, safer or more effective drugs requires a deeper understanding of the regulation of the pro-inflammatory activities of TNF and its receptors. The ubiquitously expressed TNFR1 is responsible for most TNF effects, while TNFR2 has a limited expression pattern and performs immune-regulatory functions. Despite extensive knowledge of TNFR1 signaling, the regulation of TNFR1 expression, its modifications, localization and processing are less clear and the data are scattered. Here we review the current knowledge of TNFR1 regulation and discuss the impact this has on the host.

Increased rat serum corticosterone suggests immunomodulation by stimulation of the vagal nerve

The role of the vagal nerve within the immune system has not been fully elucidated. Vagal afferents connect to several central nervous system structures, including the hypothalamus. We investigated the effect of vagal nerve stimulation (VNS) on serum corticosterone levels in rats. Corticosterone levels were measured following 1 h of high frequency (30 Hz) or low frequency (1 Hz) VNS in awake animals. There was a significant increase (p < 0.05) in serum corticosterone levels following 30 Hz VNS compared to 1 Hz VNS or sham stimulation. These results suggest an immediate effect of VNS on the hypothalamic pituitary-adrenal (HPA) axis and support the role of the vagal nerve in immunomodulation.

Glucocorticoid-induced microRNA-511 protects against TNF by down-regulating TNFR1

TNF is a central actor during inflammation and a well‐recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF‐induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1‐regulating miR‐511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR‐511 down‐regulates TNFR1 and protects against TNF, while anti‐miR‐511 up‐regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR‐511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti‐inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti‐inflammatory effects of glucocorti‐coids are mediated by induction of this miR, resulting in reduced TNFR1 expression.

Breaking the Species Barrier: Derivation of Germline‐Competent Embryonic Stem Cells from Mus spretus × C57BL/6 Hybrids

Embryonic stem (ES) cells, which can differentiate into almost all types of cells, have been derived from the house mouse Mus musculus, rat, rabbit, humans, and other species. Transmission of the genotype to the offspring of chimeras has been achieved only with M. musculus ES cells, limiting targeted mutagenesis using ES cells to this species. Mus spretus, which exhibits many genetic polymorphisms with M. musculus, displays dominant resistance to cancer and inflammation, making derived inbred strains very useful in positional cloning and interspecies mapping. We show here for the first time the derivation of ES cells from hybrid blastocysts, obtained by the mating of two different species, namely Mus musculus and Mus spretus, and their use for the generation of chimeric mice that transmit the Mus spretus genotype and phenotype to the offspring. These hybrid ES cells allow the genetic manipulation of Mus spretus, as an alternative to Mus musculus.