Interruption of neutrophil extracellular traps formation dictates host defense and tubular HOXA5 stability to augment efficacy of anti-Fn14 therapy against septic AKI.

The immunosuppressive, inflammatory microenvironment orchestrated by neutrophil extracellular traps (NETs) plays a principal role in pathogenesis of sepsis. Fibroblast growth factor-inducible molecule 14 (Fn14) has been established as a potential target for septic acute kidney injury (AKI), making further therapeutic benefits from combined NETs and Fn14 blockade possible. Methods: The concurrence of NETs and Fn14 in mice and patients with septic AKI were assessed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and in silico studies. Survival, histopathological and biochemical analyses of wild-type and PAD4-deficient CMV-Cre; PAD4 fl/fl mice with septic AKI were applied to evaluate the efficacy of either pharmacological or genetic NETs interruption in combination with Fn14 blockade. Molecular mechanisms underlying such effects were determined by CRISPR technology, fluorescence-activated cell sorter analysis (FACS), cycloheximide (CHX) pulse-chase, luciferase reporter and chromatin immunoprecipitation (ChIP) assay. Results: NETs formation is concurred with Fn14 upregulation in murine AKI models of abdominal, endotoxemic, multidrug-resistant sepsis as well as in serum samples of patients with septic AKI. Pharmacological or genetic interruption of NETs formation synergizes with ITEM-2, a monoclonal antibody (mAb) of Fn14, to prolong mice survival and provide renal protection against abdominal sepsis, the effects that could be abrogated by elimination of macrophages. Interrupting NETs formation predominantly perpetuates infiltration and survival of efferocytic growth arrest-specific protein 6+ (GAS6+) macrophages in combination with ITEM-2 therapy and enhances transcription of tubular cell-intrinsic Fn14 in a DNA methyltransferase 3a (DNMT3a)-independent manner through dismantling the proteasomes-mediated turnover of homeobox protein Hox-A5 (HOXA5) upon abdominal sepsis challenge or LPS stimuli. Pharmacological NETs interruption potentiates the anti-septic AKI efficacy of ITEM-2 in murine models of endotoxemic and multidrug-resistant sepsis. Conclusion: Our preclinical data propose that interrupting NETs formation in combination with Fn14 mAb might be a feasible therapeutic strategy for septic AKI.

TWEAKing the Hippocampus: The Effects of TWEAK on the Genomic Fabric of the Hippocampus in a Neuropsychiatric Lupus Mouse Model.

Neuropsychiatric manifestations of systemic lupus erythematosus (SLE), specifically cognitive dysfunction and mood disorders, are widely prevalent in SLE patients, and yet poorly understood. TNF-like weak inducer of apoptosis (TWEAK) has previously been implicated in the pathogenesis of neuropsychiatric lupus (NPSLE), and we have recently shown its effects on the transcriptome of the cortex of the lupus-prone mice model MRL/lpr. As the hippocampus is thought to be an important focus of NPSLE processes, we explored the TWEAK-induced transcriptional changes that occur in the hippocampus, and isolated several genes (Dnajc28, Syne2, transthyretin) and pathways (PI3K-AKT, as well as chemokine-signaling and neurotransmission pathways) that are most differentially affected by TWEAK activation. While the functional roles of these genes and pathways within NPSLE need to be further investigated, an interesting link between neuroinflammation and neurodegeneration appears to emerge, which may prove to be a promising novel direction in NPSLE research.

TWEAK and RIPK1 mediate a second wave of cell death during AKI.

Acute kidney injury (AKI) is characterized by necrotic tubular cell death and inflammation. The TWEAK/Fn14 axis is a mediator of renal injury. Diverse pathways of regulated necrosis have recently been reported to contribute to AKI, but there are ongoing discussions on the timing or molecular regulators involved. We have now explored the cell death pathways induced by TWEAK/Fn14 activation and their relevance during AKI. In cultured tubular cells, the inflammatory cytokine TWEAK induces apoptosis in a proinflammatory environment. The default inhibitor of necroptosis [necrostatin-1 (Nec-1)] was protective, while caspase inhibition switched cell death to necroptosis. Additionally, folic acid-induced AKI in mice resulted in increased expression of Fn14 and necroptosis mediators, such as receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage domain-like protein (MLKL). Targeting necroptosis with Nec-1 or by genetic RIPK3 deficiency and genetic Fn14 ablation failed to be protective at early time points (48 h). However, a persistently high cell death rate and kidney dysfunction (72-96 h) were dependent on an intact TWEAK/Fn14 axis driving necroptosis. This was prevented by Nec-1, or MLKL, or RIPK3 deficiency and by Nec-1 stable (Nec-1s) administered before or after induction of AKI. These data suggest that initial kidney damage and cell death are amplified through recruitment of inflammation-dependent necroptosis, opening a therapeutic window to treat AKI once it is established. This may be relevant for clinical AKI, since using current diagnostic criteria, severe injury had already led to loss of renal function at diagnosis.

Advanced glycation end products attenuate the function of tumor necrosis factor-like weak inducer of apoptosis to regulate the inflammatory response.

Advanced glycation end products (AGEs) are formed from the non-enzymatic glycation reaction of reducing sugars or their metabolites with the free amino groups of several biomolecules and are known to play pathophysiological roles in various inflammatory diseases. In an earlier study, it was suggested that tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has a unique role to regulate the tumor necrosis factor α (TNFα)-induced inflammatory response. In this study, we investigated the effect of the AGEs-TWEAK interaction on proinflammatory signaling responses in endothelial cells and the influence of AGEs on the cellular function of TWEAK in the inflammatory process. The effect of AGEs on the TWEAK/TNFα-induced gene expression of interleukin-8 (IL-8) was determined by real-time RT-PCR in endothelial-like EA.hy.926 cells. The pull-down assay was performed using recombinant His-tagged TWEAK and AGEs. The NF-κB activation was analyzed by Western blotting with canonical and non-canonical pathway-specific antibodies. AGEs dose-dependently inhibited TWEAK-induced IL-8 gene expression, whereas AGEs themselves had almost no effect on IL-8 expression. AGEs were found to bind directly to TWEAK in the pull-down assay. TNFα-induced IL-8 production and canonical NF-κB activation were suppressed by TWEAK pretreatment, whereas TWEAK-induced non-canonical NF-κB activation was enhanced by pretreatment. These effects induced by TWEAK pretreatment were abolished by the co-addition of AGEs. Our findings suggest that AGEs attenuate the function of TWEAK to regulate the TNFα-induced inflammatory responses, which provide important clues for understanding the significance of the AGEs-TWEAK interaction in inflammatory processes.