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Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring and loss of lung function. With limited treatment options, patients succumb to the disease within 2 to 5 years. The molecular pathogenesis of IPF regarding the immunologic changes that occur is poorly understood. We characterize a role for non-canonical aryl-hydrocarbon receptor signaling (ncAHR) in dendritic cells (DCs) that leads to production of IL-6 and increased IL-17+ cells, promoting fibrosis. TLR9 signaling in myofibroblasts is shown to regulate production of TDO2 which converts tryptophan into the endogenous AHR ligand kynurenine. Mice with augmented ncAHR signaling were created by crossing floxed AHR exon-2 deletion mice (AHRΔex2) with mice harboring a CD11c-Cre. Bleomycin (blm) was used to study fibrotic pathogenesis. Isolated CD11c+ cells and primary fibroblasts were treated ex-vivo with relevant TLR agonists and AHR modulating compounds to study how AHR signaling influenced inflammatory cytokine production. Human datasets were also interrogated. Inhibition of all AHR signaling rescued fibrosis, however, AHRΔex2 mice treated with blm developed more fibrosis and DCs from these mice were hyperinflammatory and profibrotic upon adoptive transfer. Treatment of fibrotic fibroblasts with TLR9 agonist increased expression of TDO2 and fibrotic fibroblasts activated IL-6 production in CD103+ DCs. Study of human samples corroborates the relevance of these findings in IPF patients. We also, for the first time, identify that AHR exon-2 floxed mice retain capacity for ncAHR signaling.

Patients coinfected with respiratory syncytial virus (RSV) and bacteria have longer hospital stays, higher risk of intensive care unit admission, and worse outcomes. We describe a model of RSV line 19F/methicillin-resistant Staphylococcus aureus (MRSA) USA300 coinfection that does not impair viral clearance, but prior RSV infection enhances USA300 MRSA bacterial growth in the lung. The increased bacterial burden post-RSV correlates with reduced accumulation of neutrophils and impaired bacterial killing by alveolar macrophages. Surprisingly, reduced neutrophil accumulation is likely not explained by reductions in phagocyte-recruiting chemokines or alterations in proinflammatory cytokine production compared with mice infected with S. aureus alone. Neutrophils from RSV-infected mice retain their ability to migrate toward chemokine signals, and neutrophils from the RSV-infected lung are better able to phagocytize and kill S. aureus ex vivo on a per cell basis. In contrast, while alveolar macrophages could ingest USA300 post-RSV, intracellular bacterial killing was impaired. The RSV/ S. aureus coinfected lung promotes a state of overactivation in neutrophils, demonstrated by increased production of reactive oxygen species (ROS) that can drive formation of neutrophil extracellular traps (NETs), resulting in cell death. Mice with RSV/ S. aureus coinfection had increased extracellular DNA and protein in bronchoalveolar lavage fluid and histological evidence confirmed NETosis in vivo . Taken together, these data highlight that prior RSV infection can prime the overactivation of neutrophils leading to cell death that impairs neutrophil accumulation in the lung. Additionally, alveolar macrophage killing of bacteria is impaired post-RSV. Together, these defects enhance USA300 MRSA bacterial growth in the lung post-RSV., Competing Interests: The authors declare no conflict of interest.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring and loss of lung function. With limited treatment options, patients succumb to the disease within 2-5 years. The molecular pathogenesis of IPF regarding the immunologic changes that occur is poorly understood. We characterize a role for non-canonical aryl-hydrocarbon receptor signaling (ncAHR) in dendritic cells (DCs) that leads to production of IL-6 and IL-17, promoting fibrosis. TLR9 signaling in myofibroblasts is shown to regulate production of TDO2 which converts tryptophan into the endogenous AHR ligand kynurenine. Mice with augmented ncAHR signaling were created by crossing floxed AHR exon-2 deletion mice (AHR Δex2 ) with mice harboring a CD11c-Cre. Bleomycin was used to study fibrotic pathogenesis. Isolated CD11c+ cells and primary fibroblasts were treated ex-vivo with relevant TLR agonists and AHR modulating compounds to study how AHR signaling influenced inflammatory cytokine production. Human datasets were also interrogated. Inhibition of all AHR signaling rescued fibrosis, however, AHR Δex2 mice treated with bleomycin developed more fibrosis and DCs from these mice were hyperinflammatory and profibrotic upon adoptive transfer. Treatment of fibrotic fibroblasts with TLR9 agonist increased expression of TDO2. Study of human samples corroborate the relevance of these findings in IPF patients. We also, for the first time, identify that AHR exon-2 floxed mice retain capacity for ncAHR signaling.

Coronavirus-associated coagulopathy (CAC) is a morbid and lethal sequela of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. CAC results from a perturbed balance between coagulation and fibrinolysis and occurs in conjunction with exaggerated activation of monocytes/macrophages (MO/Mφs), and the mechanisms that collectively govern this phenotype seen in CAC remain unclear. Here, using experimental models that use the murine betacoronavirus MHVA59, a well-established model of SARS-CoV-2 infection, we identify that the histone methyltransferase mixed lineage leukemia 1 (MLL1/KMT2A) is an important regulator of MO/Mφ expression of procoagulant and profibrinolytic factors such as tissue factor (F3; TF), urokinase (PLAU), and urokinase receptor (PLAUR) (herein, "coagulopathy-related factors") in noninfected and infected cells. We show that MLL1 concurrently promotes the expression of the proinflammatory cytokines while suppressing the expression of interferon alfa (IFN-α), a well-known inducer of TF and PLAUR. Using in vitro models, we identify MLL1-dependent NF-κB/RelA-mediated transcription of these coagulation-related factors and identify a context-dependent, MLL1-independent role for RelA in the expression of these factors in vivo. As functional correlates for these findings, we demonstrate that the inflammatory, procoagulant, and profibrinolytic phenotypes seen in vivo after coronavirus infection were MLL1-dependent despite blunted Ifna induction in MO/Mφs. Finally, in an analysis of SARS-CoV-2 positive human samples, we identify differential upregulation of MLL1 and coagulopathy-related factor expression and activity in CD14+ MO/Mφs relative to noninfected and healthy controls. We also observed elevated plasma PLAU and TF activity in COVID-positive samples. Collectively, these findings highlight an important role for MO/Mφ MLL1 in promoting CAC and inflammation., (© 2023 by The American Society of Hematology.)

COVID-19 induces a robust, extended inflammatory "cytokine storm" that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the cytokine storm that has been shown, in T2D, to promote excess inflammation in response to infection. Using peripheral monocytes and sera from human patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mφ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, effectively increasing inflammation. Mφs isolated from mice with a myeloid-specific deletion of SETDB2 displayed increased pathologic inflammation following coronavirus infection. Further, IFNβ directly regulates SETDB2 in Mφs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that loss of SETDB2 mediates an increased inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mφs with IFNβ reversed the inflammatory cytokine production via up-regulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mφ-mediated cytokine storm in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNβ/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Bipolar disorder (BP) is a complex psychiatric condition characterized by severe fluctuations in mood for which underlying pathological mechanisms remain unclear. Family and twin studies have identified a hereditary component to the disorder, but a single causative gene (or set of genes) has not been identified. MicroRNAs (miRNAs) are small, noncoding RNAs ∼20 nucleotides in length, that are responsible for the posttranslational regulation of multiple genes. They have been shown to play important roles in neural development as well as in the adult brain, and several miRNAs have been reported to be dysregulated in postmortem brain tissue isolated from bipolar patients. Because there are no viable cellular models to study BP, we have taken advantage of the recent discovery that somatic cells can be reprogrammed to pluripotency then directed to form the full complement of neural cells. Analysis of RNAs extracted from Control and BP patient-derived neurons identified 58 miRNAs that were differentially expressed between the two groups. Using quantitative polymerase chain reaction we validated six miRNAs that were elevated and two miRNAs that were expressed at lower levels in BP-derived neurons. Analysis of the targets of the miRNAs indicate that they may regulate a number of cellular pathways, including axon guidance, Mapk, Ras, Hippo, Neurotrophin, and Wnt signaling. Many are involved in processes previously implicated in BP, such as cell migration, axon guidance, dendrite and synapse development, and function. We have validated targets of several different miRNAs, including AXIN2 , BDNF , RELN , and ANK3 as direct targets of differentially expressed miRNAs using luciferase assays. Identification of pathways altered in patient-derived neurons suggests that disruption of these regulatory networks that may contribute to the complex phenotypes in BP.

The development of therapies for Amyotrophic Lateral Sclerosis (ALS) has been hindered by the lack of biomarkers for both identifying early disease and for monitoring the effectiveness of drugs. The identification of ALS biomarkers in presymptomatic individuals might also provide clues to the earliest biochemical correlates of the disease. Previous attempts to use plasma metabolites as biomarkers have led to contradictory results, presumably because of heterogeneity in both the underlying genetics and the disease stage in the clinical population. To eliminate these two sources of heterogeneity we have characterized plasma amino acids and other metabolites in the SOD1(G93A) transgenic mouse model for ALS. Presymptomatic SOD1(G93A) mice have significant differences in concentrations of several plasma metabolites compared to wild type animals, most notably in the concentrations of aspartate, cystine/cysteine, and phosphoethanolamine, and in changes indicative of methylation defects. There are significant changes in amino acid compositions between 50 and 70days of age in both the SOD1(G93A) and wild type mice, and several of the age-related and disease-related differences in metabolite concentration were also gender-specific. Many of the SOD1(G93A)-related differences could be altered by treatment of mice with methionine sulfoximine, which extends the lifespan of this mouse, inhibits glutamine synthetase, and modifies brain methylation reactions. These studies show that assaying plasma metabolites can effectively distinguish transgenic mice from wild type, suggesting that one or more plasma metabolites might be useful biomarkers for the disease in humans, especially if genetic and longitudinal analysis is used to reduce population heterogeneity., (© 2013.)

Objective: To investigate the role of sex and the role of ammonia and amino acid metabolism, specifically the activity of glutamine synthetase, in survival and disease progression in amyotrophic lateral sclerosis., Methods: We tested treatment with methionine sulfoximine (MSO) on the lifespan and neuromuscular ability of male and female SOD1 mice as measured by their ability to maintain their grip on an inverted wire grid. We also tested the effects of castration and ovariectomization on those measurements., Results: MSO treatment improves the survival of both male and female mice, but the effects are significantly greater on female mice. Saline-treated (control) female mice have delayed neuromuscular degeneration compared with saline-treated male mice, and MSO further delays disease progression in females, to a greater extent than in males. Ovariectomization or castration completely eliminates the effect of the drug on either survival or neuromuscular deterioration., Conclusions: Sex is an important factor in disease progression and the response of SOD1 mice to a drug targeting a central enzyme in nitrogen metabolism, with female sex hormones playing a greater role than male sex hormones. Glutamine synthetase, or its reactants and products, therefore plays a role in this disease, and the sex specificity of treatments aimed at this or other metabolic targets may therefore be an important factor in the development of therapies to treat amyotrophic lateral sclerosis., (Copyright © 2012 Elsevier HS Journals, Inc. All rights reserved.)

In an effort to alter the levels of neurochemicals involved in excitotoxicity, we treated mice with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. Since glutamate toxicity has been proposed as a mechanism for the degeneration of motor neurons in a variety of neurodegenerative diseases, we tested the effects of MSO on the transgenic mouse that overexpresses the mutant human SOD1(G93A) gene, an animal model for the primary inherited form of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). This treatment in vivo reduced glutamine synthetase activity measured in vitro by 85%. Proton magnetic resonance spectroscopy, with magic angle spinning of intact samples of brain tissue, showed that MSO treatment reduced brain levels of glutamine by 60% and of glutamate by 30% in both the motor cortex and the anterior striatum, while also affecting levels of GABA and glutathione. Kaplan-Meyer survival analysis revealed that MSO treatment significantly extended the lifespan of these mice by 8% (p<0.01). These results show that in the SOD1(G93A) model of neurodegenerative diseases, the concentration of brain glutamate (determined with (1)H-MRS) can be lowered by inhibiting in vivo the synthesis of glutamine with non-toxic doses of MSO.

The multifunctional protein CAD initiates de novo pyrimidine biosynthesis in mammalian cells. CAD is activated by MAP kinase (Erk1/2) just prior to the S phase of the cell cycle, when the demand for pyrimidine nucleotides is greatest, and down-regulated as the cells emerge from S phase by protein kinase A (PKA) phosphorylation. MAP kinase phosphorylates Thr456, while PKA phosphorylates Ser1406 and Ser1859, although only Ser1406 is involved in regulation. LC/mass spectrometry showed that Ser1873, a residue that lies within a putative protein kinase C (PKC) consensus sequence is also phosphorylated. Purified CAD was reacted with ATP and a panel of eight PKC isozymes. Most isozymes resulted in limited CAD phosphorylation, but the delta and epsilon isozymes were most effective. While the level of Thr456 phosphorylation is very low in confluent cells, exposure of stationary BHK 165-23 cells to the PKC activator, phorbol 12-myristate-13-acetate (PMA) resulted in a 3-fold increase in the modification of this residue. The stimulation of Thr456 phosphorylation was blocked by PKC inhibitors. The PKA inhibitor, H-89, also stimulated PMA-induced Thr456 modification probably because PKA mediated phosphorylation of CAD Ser1406 antagonizes the MAP kinase phosphorylation. Thus, the extent of Thr456 phosphorylation and the activation of pyrimidine biosynthesis depend on the synergistic and antagonistic interactions of three signaling pathways, MAP kinase, PKC and PKA. Deletions mutants lacking the putative PKC site, Ser1873 do not exhibit PMA induced Thr456 phosphorylation. We conclude that the activating MAP kinase phosphorylation of CAD proceeds through a PKC dependent pathway.