Two-pore potassium channel TREK-1 (K2P2.1) regulates NLRP3 inflammasome activity in macrophages.

Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1-/- mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1ß, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescence-based assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1ß secretion in wt AMs, whereas activation was significantly reduced in TREK-1-/- AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1-/- BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significantly lower compared with TREK-1-/- BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1-/- BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1-/- BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1-/- mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.

Deficiency of Acute-Phase Serum Amyloid A Exacerbates Sepsis-Induced Mortality and Lung Injury in Mice.

Serum amyloid A (SAA) is a family of proteins, the plasma levels of which may increase >1000-fold in acute inflammatory states. We investigated the role of SAA in sepsis using mice deficient in all three acute-phase SAA isoforms (SAA-TKO). SAA deficiency significantly increased mortality rates in the three experimental sepsis mouse models: cecal ligation and puncture (CLP), cecal slurry (CS) injection, and lipopolysaccharide (LPS) treatments. SAA-TKO mice had exacerbated lung pathology compared to wild-type (WT) mice after CLP. A bulk RNA sequencing performed on lung tissues excised 24 h after CLP indicated significant enrichment in the expression of genes associated with chemokine production, chemokine and cytokine-mediated signaling, neutrophil chemotaxis, and neutrophil migration in SAA-TKO compared to WT mice. Consistently, myeloperoxidase activity and neutrophil counts were significantly increased in the lungs of septic SAA-TKO mice compared to WT mice. The in vitro treatment of HL-60, neutrophil-like cells, with SAA or SAA bound to a high-density lipoprotein (SAA-HDL), significantly decreased cellular transmigration through laminin-coated membranes compared to untreated cells. Thus, SAA potentially prevents neutrophil transmigration into injured lungs, thus reducing exacerbated tissue injury and mortality. In conclusion, we demonstrate for the first time that endogenous SAA plays a protective role in sepsis, including ameliorating lung injury.

ASK1 Regulates Bleomycin-induced Pulmonary Fibrosis.

Pulmonary fibrosis (PF) is an abnormal remodeling of cellular composition and extracellular matrix that results in histological and functional alterations in the lungs. Apoptosis signal-regulating kinase-1 (ASK1) is a member of the mitogen-activated protein (MAP) kinase family that is activated by oxidative stress and promotes inflammation and apoptosis. Here we show that bleomycin-induced PF is reduced in Ask1 knockout mice (Ask1-/-) compared with wild-type (WT) mice, with improved survival and histological and functional parameters restored to basal levels. In WT mice, bleomycin caused activation of ASK1, p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) in lung tissue, as well as changes in redox indicators (thioredoxin and heme-oxygenase-1), collagen content, and epithelial-mesenchymal transition markers (EMTs). These changes were largely restored toward untreated WT control levels in bleomycin-treated Ask1-/- mice. We further investigated whether treatment of WT mice with an ASK1 inhibitor, selonsertib (GS-4997), during the fibrotic phase would attenuate the development of PF. We found that pharmacological inhibition of ASK1 reduced activation of ASK1, p38, and ERK1/2 and promoted the restoration of redox and EMT indicators, as well as improvements in histological parameters. Our results suggest that ASK1 plays a central role in the development of bleomycin-induced PF in mice via p38 and ERK1/2 signaling. Together, these data indicate a possible therapeutic target for PF that involves an ASK1/p38/ERK1/2 axis.

BDNF deficiency and enriched environment treatment affect neurotransmitter gene expression differently across ages.

Deficiency of activity-induced expression of brain-derived neurotrophic factor (BDNF) disturbs neurotransmitter gene expression. Enriched environment treatment (EET) ameliorates the defects. However, how BDNF deficiency and EET affect the neurotransmitter gene expression differently across ages remains unclear. We addressed this question by determining the neurotransmitter gene expression across three life stages in wild-type and activity-dependent BDNF-deficient (KIV) mice. Mice received 2-months of standard control treatment (SCT) or EET at early-life development (ED: 0-2 months), young adulthood (2-4 months), and old adulthood (12-14 months) (N = 16/group). Half of these mice received additional 1-month SCT to examine persisting EET effects. High-throughput quantitative reverse transcription polymerase chain reaction measured expression of 81 genes for dopamine, adrenaline, serotonin, gamma aminobutyric acid, glutamate, acetylcholine, and BDNF systems in the frontal cortex (FC) and hippocampus. Results revealed that BDNF deficiency mostly reduced neurotransmitter gene expression, greatest at ED in the FC. EET increased expression of a larger number of genes at ED than adulthood, particularly in the KIV FC. Many genes down-regulated in KIV mice were up-regulated by EET, which persisted when EET was provided at ED (e.g., 5-hydroxytryptamine (serotonin) transporter [5HTT], ADRA1D, GRIA3, GABRA5, GABBR2). In both the regions, BDNF deficiency decreased the density of gene co-expression network specifically at ED, while EET increased the density and hub genes (e.g., GAT1, GABRG3, GRIN1, CHRNA7). These results suggest that BDNF deficiency, which occurs under chronic stress, causes neurotransmitter dysregulations prominently at ED, particularly in the FC. EET at ED may be most effective to normalize the dysregulations, providing persisting effects later in life. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Science badges can be found at https://cos.io/our-services/open-science-badges/.

Apoptosis signal-regulating kinase-1 promotes inflammasome priming in macrophages.

We previously showed that mice deficient in apoptosis signal-regulating kinase-1 (ASK1) were partially protected against ventilator-induced lung injury. Because ASK1 can promote both cell death and inflammation, we hypothesized that ASK1 activation regulates inflammasome-mediated inflammation. Mice deficient in ASK1 expression (ASK1-/-) exhibited significantly less inflammation and lung injury (as measured by neutrophil infiltration, IL-6, and IL-1ß) in response to treatment with inhaled lipopolysaccharide (LPS) compared with wild-type (WT) mice. To determine whether this proinflammatory response was mediated by ASK1, we investigated inflammasome-mediated responses to LPS in primary macrophages and bone marrow-derived macrophages (BMDMs) from WT and ASK1-/- mice, as well as the mouse alveolar macrophage cell line MH-S. Cells were treated with LPS alone for priming or LPS followed by ATP for activation. When macrophages were stimulated with LPS followed by ATP to activate the inflammasome, we found a significant increase in secreted IL-1ß from WT cells compared with ASK1-deficient cells. LPS priming stimulated an increase in NOD-like receptor 3 (NLRP3) and pro-IL-1ß in WT BMDMs, but expression of NLRP3 was significantly decreased in ASK1-/- BMDMs. Subsequent ATP treatment stimulated an increase in cleaved caspase-1 and IL-1ß in WT BMDMs compared with ASK1-/- BMDMs. Similarly, treatment of MH-S cells with LPS + ATP caused an increase in both cleaved caspase-1 and IL-1ß that was diminished by the ASK-1 inhibitor NQDI1. These results demonstrate, for the first time, that ASK1 promotes inflammasome priming.

Effects of antidepressant treatment on mice lacking brain-derived neurotrophic factor expression through promoter IV.

Brain-derived neurotrophic factor (BDNF) is implicated in the pathophysiology of major depression; mice lacking BDNF expression through promoter IV (BDNF-KIV) exhibit a depression-like phenotype. We tested our hypothesis that deficits caused by promoter IV deficiency (depression-like behavior, decreased levels of BDNF, and neurogenesis in the hippocampus) could be rescued by a 3-week treatment with different types of antidepressants: fluoxetine, phenelzine, duloxetine, or imipramine. Each antidepressant reduced immobility time in the tail suspension test without affecting locomotor activity in the open field test in both BDNF-KIV and control wild type mice, except that phenelzine increased locomotor activity in wild type mice and anxiety-like behavior in BDNF-KIV mice. The antidepressant treatments were insufficient to reverse decreased BDNF levels caused by promoter IV deficiency. No antidepressant treatment increased the hippocampal progenitors of either genotype, whereas phenelzine decreased the surviving progenitors in both genotypes. The antidepressant treatments differently affected the dendritic extension of hippocampal immature neurons: fluoxetine and imipramine increased extension in both genotypes, duloxetine increased it only in BDNF-KIV mice, and phenelzine decreased it only in wild type mice. Interestingly, a saline-only injection increased neurogenesis and dendrite extensions in both genotypes. Our results indicate that the behavioral effects in the tail suspension test by antidepressants do not require promoter IV-driven BDNF expression and occur without a detectable increase in hippocampal BDNF levels and neurogenesis but may involve increased dendritic reorganisation of immature neurons. In conclusion, the antidepressant treatment demonstrated limited efficacy; it partially reversed the defective phenotypes caused by promoter IV deficiency but not hippocampal BDNF levels.

Cervical spinal cord injury leads to injury and altered metabolism in the lungs.

High-cervical spinal cord injury often disrupts respiratory motor pathways and disables breathing in the affected population. Moreover, cervically injured individuals are at risk for developing acute lung injury, which predicts substantial mortality rates. While the correlation between acute lung injury and spinal cord injury has been found in the clinical setting, the field lacks an animal model to interrogate the fundamental biology of this relationship. To begin to address this gap in knowledge, we performed an experimental cervical spinal cord injury (N = 18) alongside sham injury (N = 3) and naïve animals (N = 15) to assess lung injury in adult rats. We demonstrate that animals display some early signs of lung injury two weeks post-spinal cord injury. While no obvious histological signs of injury were observed, the spinal cord injured cohort displayed significant signs of metabolic dysregulation in multiple pathways that include amino acid metabolism, lipid metabolism, and N-linked glycosylation. Collectively, we establish for the first time a model of lung injury after spinal cord injury at an acute time point that can be used to monitor the progression of lung damage, as well as identify potential targets to ameliorate acute lung injury.

Targeting the Microbiome to Improve Gut Health and Breathing Function After Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition characterized by impaired motor and sensory function, as well as internal organ pathology and dysfunction. This internal organ dysfunction, particularly gastrointestinal (GI) complications, and neurogenic bowel, can reduce the quality of life of individuals with an SCI and potentially hinder their recovery. The gut microbiome impacts various central nervous system functions and has been linked to a number of health and disease states. An imbalance of the gut microbiome, i.e., gut dysbiosis, contributes to neurological disease and may influence recovery and repair processes after SCI. Here we examine the impact of high cervical SCI on the gut microbiome and find that transient gut dysbiosis with persistent gut pathology develops after SCI. Importantly, probiotic treatment improves gut health and respiratory motor function measured through whole-body plethysmography. Concurrent with these improvements was a systemic decrease in the cytokine tumor necrosis factor-alpha and an increase in neurite sprouting and regenerative potential of neurons. Collectively, these data reveal the gut microbiome as an important therapeutic target to improve visceral organ health and respiratory motor recovery after SCI. Research Highlights: Cervical spinal cord injury (SCI) causes transient gut dysbiosis and persistent gastrointestinal (GI) pathology.Treatment with probiotics after SCI leads to a healthier GI tract and improved respiratory motor recovery.Probiotic treatment decreases systemic tumor necrosis factor-alpha and increases the potential for sprouting and regeneration of neurons after SCI.The gut microbiome is a valid target to improve motor function and secondary visceral health after SCI.

Spatial metabolomics reveals glycogen as an actionable target for pulmonary fibrosis.

Matrix assisted laser desorption/ionization imaging has greatly improved our understanding of spatial biology, however a robust bioinformatic pipeline for data analysis is lacking. Here, we demonstrate the application of high-dimensionality reduction/spatial clustering and histopathological annotation of matrix assisted laser desorption/ionization imaging datasets to assess tissue metabolic heterogeneity in human lung diseases. Using metabolic features identified from this pipeline, we hypothesize that metabolic channeling between glycogen and N-linked glycans is a critical metabolic process favoring pulmonary fibrosis progression. To test our hypothesis, we induced pulmonary fibrosis in two different mouse models with lysosomal glycogen utilization deficiency. Both mouse models displayed blunted N-linked glycan levels and nearly 90% reduction in endpoint fibrosis when compared to WT animals. Collectively, we provide conclusive evidence that lysosomal utilization of glycogen is required for pulmonary fibrosis progression. In summary, our study provides a roadmap to leverage spatial metabolomics to understand foundational biology in pulmonary diseases.

SARS-CoV-2 Spike Protein Regulation of Angiotensin Converting Enzyme 2 and Tissue Renin-Angiotensin Systems: Influence of Biologic Sex.

Angiotensin converting enzyme 2 (ACE2) is an enzyme that limits activity of the renin-angiotensin system (RAS) and also serves as a receptor for the SARS-CoV-2 Spike (S) protein. Binding of S protein to ACE2 causes internalization which activates local RAS. ACE2 is on the X chromosome and its expression is regulated by sex hormones. In this study, we defined ACE2 mRNA abundance and examined effects of S protein on ACE2 activity and/or angiotensin II (AngII) levels in pivotal tissues (lung, adipose) from male and female mice. In lung, ACE2 mRNA abundance was reduced following gonadectomy (GDX) of male and female mice and was higher in XX than XY mice of the Four Core Genotypes (FCG). Reductions in lung ACE2 mRNA abundance by GDX occurred in XX, but not XY FCG female mice. Lung mRNA abundance of ADAM17 and TMPRSS2, enzymes that shed cell surface ACE2 and facilitate viral cell entry, was reduced by GDX in male but not female mice. For comparison, adipose ACE2 mRNA abundance was higher in female than male mice and higher in XX than XY FCG mice. Adipose ADAM17 mRNA abundance was increased by GDX of male and female mice. S protein reduced ACE2 activity in alveolar type II epithelial cells and 3T3-L1 adipocytes. Administration of S protein to male and female mice increased lung AngII levels and decreased adipose ACE2 activity in male but not female mice. These results demonstrate that sex differences in ACE2 expression levels may impact local RAS following S protein exposures.

Polycystin-1 interacts with TAZ to stimulate osteoblastogenesis and inhibit adipogenesis.

The molecular mechanisms that transduce the osteoblast response to physical forces in the bone microenvironment are poorly understood. Here, we used genetic and pharmacological experiments to determine whether the polycystins PC1 and PC2 (encoded by Pkd1 and Pkd2) and the transcriptional coactivator TAZ form a mechanosensing complex in osteoblasts. Compound-heterozygous mice lacking 1 copy of Pkd1 and Taz exhibited additive decrements in bone mass, impaired osteoblast-mediated bone formation, and enhanced bone marrow fat accumulation. Bone marrow stromal cells and osteoblasts derived from these mice showed impaired osteoblastogenesis and enhanced adipogenesis. Increased extracellular matrix stiffness and application of mechanical stretch to multipotent mesenchymal cells stimulated the nuclear translocation of the PC1 C-terminal tail/TAZ (PC1-CTT/TAZ) complex, leading to increased runt-related transcription factor 2-mediated (Runx2-mediated) osteogenic and decreased PPARγ-dependent adipogenic gene expression. Using structure-based virtual screening, we identified a compound predicted to bind to PC2 in the PC1:PC2 C-terminal tail region with helix:helix interaction. This molecule stimulated polycystin- and TAZ-dependent osteoblastogenesis and inhibited adipogenesis. Thus, we show that polycystins and TAZ integrate at the molecular level to reciprocally regulate osteoblast and adipocyte differentiation, indicating that the polycystins/TAZ complex may be a potential therapeutic target to increase bone mass.

Activity-Based Proteomic Profiling of Deubiquitinating Enzymes in Salmonella-Infected Macrophages Leads to Identification of Putative Function of UCH-L5 in Inflammasome Regulation.

Although protein ubiquitination has been shown to regulate multiple processes during host response to Salmonella enterica serovar Typhimurium infection, specific functions of host deubiquitinating enzymes remain unknown in this bacterial infection. By using chemical proteomics approach, in which deubiquitinating enzymes were labeled by an active-site probe and analyzed by quantitative proteomics, we identified novel deubiquitinases in chicken macrophages based on their reactivity with the probe. Also, we detected down-regulation of UCH-L3, and USP4 as well as up-regulation of USP5 and UCH-L5 deubiquitinating enzymes in macrophages infected with Salmonella Typhimurium. We showed that decrease in either UCH-L5 activity, or in UCH-L5 protein amount in chicken and human macrophages infected or stimulated with LPS/nigericin, led to decreased IL-1ß release. These data point towards a putative role of UCH-L5 in inflammasome regulation during Salmonella infection. Because inflammasome activation is important in innate resistance to these bacteria, one would expect that naturally occurring or therapeutically induced alteration in UCH-L5 activation would influence disease outcome and could represent a target for new therapeutic approaches.