p53-Independent Roles of MDM2 in NF-κB Signaling: Implications for Cancer Therapy, Wound Healing, and Autoimmune Diseases

Murine double minute-2 (MDM2) is an intracellular molecule with multiple biologic functions. It serves as a negative regulator of p53 and thereby limits cell cycle arrest and apoptosis. Because MDM2 blockade suppresses tumor cell growth in vitro and in vivo, respective MDM2 inhibition is currently evaluated as anti-cancer therapy in clinical trials. However, the anti-proliferative effects of MDM2 inhibition also impair regenerative cell growth upon tissue injury. This was so far documented for tubular repair upon postischemic acute kidney injury and might apply to wound healing responses in general. Furthermore, MDM2 has numerous p53-independent effects. As a new entry, MDM2 was identified to act as a co-transcription factor for nuclear factor-kappa-light-enhancer of activated B cells (NF-κB) at cytokine promoters. This explains the potent anti-inflammatory effects of MDM2 inhibitors in vitro and in vivo. For example, the NF-κB-antagonistic and p53-agonistic activities of MDM2 inhibitors elicit potent therapeutic effects on experimental lymphoproliferative autoimmune disorders such as systemic lupus erythematosus. In this review, we discuss the classic p53-dependent, the recently discovered p53-independent, and the NF-κB-agonistic biologic functions of MDM2. We describe its complex regulatory role on p53 and NF-κB signaling and name areas of research that may help to foresee previously unexpected effects or potential alternative indications of therapeutic MDM2 blockade

A guide to crystal‐related and nano‐ or microparticle‐related tissue responses

Crystals and nano‐ and microparticles form inside the human body from intrinsic proteins, minerals, or metabolites or enter the body as particulate matter from occupational and environmental sources. Associated tissue injuries and diseases mostly develop from cellular responses to such crystal deposits and include inflammation, cell necrosis, granuloma formation, tissue fibrosis, and stone‐related obstruction of excretory organs. But how do crystals and nano‐ and microparticles trigger these biological processes? Which pathomechanisms are identical across different particle types, sizes, and shapes? In addition, which mechanisms are specific to the atomic or molecular structure of crystals or to specific sizes or shapes? Do specific cellular or molecular mechanisms qualify as target for therapeutic interventions? Here, we provide a guide to approach this diverse and multidisciplinary research domain. We give an overview about the clinical spectrum of crystallopathies, about shared and specific pathomechanisms as a conceptual overview before digging deeper into the specialty field of interest

The Immune System in Tissue Environments Regaining Homeostasis after Injury: Is "Inflammation" Always Inflammation?

Inflammation is a response to infections or tissue injuries. Inflammation was once defined by clinical signs, later by the presence of leukocytes, and nowadays by expression of "proinflammatory" cytokines and chemokines. But leukocytes and cytokines often have rather anti-inflammatory, proregenerative, and homeostatic effects. Is there a need to redefine "inflammation"? In this review, we discuss the functions of "inflammatory" mediators/regulators of the innate immune system that determine tissue environments to fulfill the need of the tissue while regaining homeostasis after injury

The Immune System in Tissue Environments Regaining Homeostasis after Injury: Is “Inflammation” Always Inflammation?

Inflammation is a response to infections or tissue injuries. Inflammation was once defined by clinical signs, later by the presence of leukocytes, and nowadays by expression of “proinflammatory” cytokines and chemokines. But leukocytes and cytokines often have rather anti-inflammatory, proregenerative, and homeostatic effects. Is there a need to redefine “inflammation”? In this review, we discuss the functions of “inflammatory” mediators/regulators of the innate immune system that determine tissue environments to fulfill the need of the tissue while regaining homeostasis after injury

Regulated necrosis-related molecule mRNA expression in humans and mice and in murine acute tissue injury and systemic autoimmunity leading to progressive organ damage, and progressive fibrosis

The species-specific, as well as organ-specific expression of regulated necrosis (RN)-related molecules, is not known. We determined the expression levels of tumour necrosis factor receptor-1 (TNFR1), receptor activated protein kinase (RIPK) 1, RIPK3, mixed lineage kinase domain-like (MLKL), CASP8, Fas-associated protein with death domain (FADD), cellular inhibitor of apoptosis protein (CIAP) 1, CIAP2, glutathione peroxidase-4 (GPX4), cyclophilin D (CYPD), CASP1, NLRP3 and poly(ADP-ribose) polymerase-1 (PARP1) in human and mouse solid organs. We observed significant differences in expression of these molecules between human and mice. In addition, we characterized their expression profiles in acute as well as persistent tissue injury and chronic tissue remodelling using acute and chronic kidney injury models. We observed that the degree and pattern of induction of RN-related molecules were highly dependent on the trigger and disease pathogenesis. Furthermore, we studied their expression patterns in mice with lupus-like systemic autoimmunity, which revealed that the expression of MLKL, GPX4 and PARP1 significantly increased in the spleen along disease progression and CASP1, RIPK1, RIPK3 and CYPD were higher at the earlier stages but were significantly decreased in the later stages. In contrast, in the kidney, the expression of genes involved in pyroptosis, e. g. NLRP3 and CASP1 were significantly increased and TNFR1, RIPK1, RIPK3, CIAP1/2 and GPX4 were significantly decreased along the progression of lupus nephritis (LN). Thus, the organ-and species-specific expression of RN-related molecules should be considered during designing experiments, interpreting the results as well as extrapolating the conclusions from one species or organ to another species or organ respectively

The Long Pentraxin PTX3 Is an Endogenous Inhibitor of Hyperoxaluria-Related Nephrocalcinosis and Chronic Kidney Disease

The long pentraxin 3 (PTX3) exerts a variety of regulatory functions in acute and chronic tissue inflammation. In particular, PTX3 acts as an opsonin for a variety of pathogens and endogenous particles. We hypothesized that PTX3 would exhibit opsonin-like functions toward calcium oxalate crystals, too, and inhibit crystal growth. This process is fundamental in kidney stone disease as well as in hyperoxaluria-related nephrocalcinosis, the paradigmatic cause of chronic kidney disease (CKD) in children with primary hyperoxaluria type I due to genetic defects in oxalate metabolism. Direct effects of PTX3 on calcium oxalate crystals were investigated in chemico by adding recombinant PTX3 to supersaturated calcium and oxalate solutions. PTX3, but not isomolar concentrations of albumin, dose-dependently inhibited crystal growth. In vivo, the PTX3 protein was undetectable in tubular epithelial cells and urine of wild-type mice under physiological conditions. However, its levels increased within 3 weeks of feeding an oxalate-rich diet, an exposure inducing hyperoxaluria-related nephrocalcinosis and CKD in selected mouse strains (male and female C57BL/6N and male Balb/c mice) but not in others (male and female 129SV and CD-1, male and female Balb/c mice). Genetic ablation of ptx3 in nephrocalcinosis un-susceptible B6;129 mice was sufficient to raise the oxalate nephropathy phenotype observed in susceptible strains. We conclude that PTX3 is an endogenous inhibitor of calcium oxalate crystal growth. This mechanism limits hyperoxaluria-related nephrocalcinosis, e.g., in primary or secondary hyperoxaluria, and potentially also in the more prevalent kidney stone disease

Cathepsin S inhibition suppresses systemic lupus erythematosus and lupus nephritis because cathepsin S is essential for MHC class II-mediated CD4 T cell and B cell priming

Objectives: Major histocompatibility complex (MHC) class II-mediated priming of T and B lymphocytes is a central element of autoimmunity in systemic lupus erythematosus (SLE) and lupus nephritis. The cysteine protease cathepsin S degrades the invariant peptide chain during MHC II assembly with antigenic peptide in antigen-presenting cells; therefore, we hypothesised that cathepsin S inhibition would be therapeutic in SLE. Methods: We developed a highly specific small molecule, orally available, cathepsin S antagonist, RO5461111, with suitable pharmacodynamic and pharmacokinetic properties that efficiently suppressed antigen-specific T cell and B cell priming in vitro and in vivo. Results: When given to MRL-Fas(lpr) mice with SLE and lupus nephritis, RO5461111 significantly reduced the activation of spleen dendritic cells and the subsequent expansion and activation of CD4 T cells and CD4/CD8 double-negative T cells. Cathepsin S inhibition impaired the spatial organisation of germinal centres, suppressed follicular B cell maturation to plasma cells and Ig class switch. This reversed hypergammaglobulinemia and significantly suppressed the plasma levels of numerous IgG (but not IgM) autoantibodies below baseline, including anti-dsDNA. This effect was associated with less glomerular IgG deposits, which protected kidneys from lupus nephritis. Conclusions: Together, cathepsin S promotes SLE by driving MHC class II-mediated T and B cell priming, germinal centre formation and B cell maturation towards plasma cells. These afferent immune pathways can be specifically reversed with the cathepsin S antagonist RO5461111, which prevents lupus nephritis progression even when given after disease onset. This novel therapeutic strategy could correct a common pathomechanism of SLE and other immune complex-related autoimmune diseases

Activated platelets induce MLKL-driven neutrophil necroptosis and release of neutrophil extracellular traps in venous thrombosis

Venous thromboembolic (VIE) disease, often manifesting as deep vein thrombosis or pulmonary embolism, involves clot formation consisting of blood cells and platelets locked in plasma protein and chromatin networks. The latter derives from neutrophil extracellular traps released by dying neutrophils;however, the molecular mechanisms of neutrophil death in VIE remains unknown. We speculated that mixed lineage kinase-like (MLKL)-driven neutrophil necroptosis contributes to VTE. Indeed, human inferior venous cava thrombus material stained positive for phosphorylated MLKL, the activated version of MLKL that executes necroptotic cell death. In mice, MLKL immunostaining showed co-localization of MLKL with citrullinated histone H3, a marker of neutrophil extracellular trap (NET) formation. These data provide indirect support for a role of MLKL-mediated necroptosis. As a functional proof, both the stabilizer of receptor-interacting protein kinase-1 (RIPK1) and necroptosis inhibitor necrostatin-1s as well as genetic deficiency of MLKL partially prevented clot formation upon inferior vena cava ligation in mice. In both experiments terminal deoxynucleotidyl transferase dUTP nick-end labeling, RIPK3, and citrullinated histone H3+ areas were markedly reduced within the remnant thrombus. In vitro, thrombin-activated platelets induced cell death and NET formation in human neutrophils, which was inhibited by necrostatin-1s treatment. Necrostatin-1s and necrosulfonamide also inhibited neutrophil-platelet aggregate formation induced by tumor necrosis factor-a but had no effect on platelet activation itself. We conclude that in VTE, activated platelets, and possibly other triggers, induce neutrophil necroptosis, a process contributing to clot formation by releasing chromatin in the extracellular space