ABSTRACT
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.
Subject(s)
Inflammasomes , Potassium Channels, Tandem Pore Domain , Tetrahydronaphthalenes , Tetrazoles , Animals , Mice , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Potassium/metabolism , Lipopolysaccharides/pharmacology , Lipopolysaccharides/metabolism , Mice, Knockout , Potassium Channels, Tandem Pore Domain/genetics , Potassium Channels, Tandem Pore Domain/metabolism , Macrophages/metabolism , Caspase 1/metabolism , Adenosine Triphosphate/pharmacology , Adenosine Triphosphate/metabolism , Interleukin-1beta/metabolismABSTRACT
Venezuelan equine encephalitis virus (VEEV) causes a febrile illness that can progress to neurological disease with the possibility of death in human cases. The evaluation and optimization of therapeutics that target brain infections demands knowledge of the host's response to VEEV, the dynamics of infection, and the potential for within-host evolution of the virus. We hypothesized that selective pressures during infection of the brain may differ temporally and spatially and so we investigated the dynamics of the host response, viral transcript levels, and genetic variation of VEEV TC-83 in eight areas of the brain in mice over 7 days post-infection (dpi). Viral replication increased throughout the brain until 5-6 dpi and decreased thereafter with neurons as the main site of viral replication. Low levels of genetic diversity were noted on 1 dpi and were followed by an expansion in the genetic diversity of VEEV and nonsynonymous (Ns) mutations that peaked by 5 dpi. The pro-inflammatory response and the influx of immune cells mirrored the levels of virus and correlated with substantial damage to neurons by 5 dpi and increased activation of microglial cells and astrocytes. The prevalence and dynamics of Ns mutations suggest that the VEEV is under selection within the brain and that progressive neuroinflammation may play a role in acting as a selective pressure. IMPORTANCE Treatment of encephalitis in humans caused by Venezuelan equine encephalitis virus (VEEV) from natural or aerosol exposure is not available, and hence, there is a great interest to address this gap. In contrast to natural infections, therapeutic treatment of infections from aerosol exposure will require fast-acting drugs that rapidly penetrate the blood-brain barrier, engage sites of infection in the brain and mitigate the emergence of drug resistance. Therefore, it is important to understand not only VEEV pathogenesis, but the trafficking of the viral population within the brain, the potential for within-host evolution of the virus, and how VEEV might evolve resistance.
Subject(s)
Encephalitis Virus, Venezuelan Equine , Encephalitis , Animals , Humans , Mice , Brain , Cell Death , Encephalitis Virus, Venezuelan Equine/genetics , Genetic Variation , Encephalitis/virologyABSTRACT
Two siblings with CF are homozygous for F508del (referred to as Subject A and Subject B). Despite having the same CFTR genotype and similar environment, these two subjects exhibited different disease phenotypes. We analyzed their medical records and CF Foundation Registry data and measured inflammatory protein mediators in their sputum samples. Then, we examined the longitudinal relationships between inflammatory markers and disease severity for each subject and compared between them. Subject A presented a more severe disease than Subject B. During the study period, Subject A had two pulmonary exacerbations (PEs) whereas Subject B had one mild PE. The forced expiratory volume in 1 s (FEV1, % predicted) values for Subject A were between 34-45% whereas for Subject B varied between 48-90%. Inflammatory protein mediators associated with neutrophils, Th1, Th2, and Th17 responses were elevated in sputum of Subject A compared with Subject B, and also in samples collected prior to and during PEs for both subjects. Neutrophilic elastase (NE) seemed to be the most informative biomarkers. The infectious burden between these two subjects was different.
Subject(s)
Amino Acid Sequence , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis , Homozygote , Inflammation Mediators/metabolism , Sequence Deletion , Siblings , T-Lymphocytes, Helper-Inducer/metabolism , Biomarkers/metabolism , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Female , Humans , Leukocyte Elastase/metabolism , Male , Sputum/metabolismABSTRACT
Hypersensitivity pneumonitis (HP) is an interstitial lung disease that may progress to fibrosis and significant risk of death. HP develops following repeated exposures to inhaled environmental antigens; however, only a fraction of the exposed population develops the disease, suggesting that host genetics contribute to disease susceptibility. We used the BXD family of mice with the Saccharopolyspora rectivirgula (SR) model of HP to investigate the role of genetics in susceptibility to HP. The BXD family is derived from a B6 mother and a D2 father and has been used to map susceptibility loci to numerous diseases. B6, D2, and BXD progeny strains were exposed to SR for 3 wk, and the development of HP was monitored. The B6 and D2 strains developed alveolitis; however, the cellular composition was neutrophilic in the D2 strain and more lymphocytic in the B6 strain. Hematoxylin-eosin staining of lung sections revealed lymphoid aggregates in B6 lungs, whereas D2 lungs exhibited a neutrophilic infiltration. Twenty-eight BXD strains of mice were tested, and the results reveal significant heritable variation for numbers of CD4+ or CD8+ T cells in the air spaces. There was significant genetic variability for lymphoid aggregates and alveolar wall thickening. We mapped a significant quantitative trait locus (QTL) on chromosome 18 for CD8+CD69+ T cells that includes cadherin 2 (Cdh2), an excellent candidate gene associated with epithelial-mesenchymal transition, which is upregulated in lungs of strains with HP. These results demonstrate that the BXD family is a valuable and translationally relevant model to identify genes contributing to HP and to devise early and effective interventions.
Subject(s)
Alveolitis, Extrinsic Allergic/genetics , Alveolitis, Extrinsic Allergic/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Genetic Variation/genetics , Alveolitis, Extrinsic Allergic/microbiology , Animals , Epithelial-Mesenchymal Transition/genetics , Female , Lung/immunology , Lung/microbiology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Neutrophils/immunology , Saccharopolyspora/immunology , Up-Regulation/geneticsABSTRACT
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.
Subject(s)
Apoptosis/drug effects , Inflammasomes/drug effects , MAP Kinase Kinase Kinase 5/metabolism , Macrophages/drug effects , Animals , Carrier Proteins/metabolism , Cell Line , Inflammasomes/metabolism , Inflammation/drug therapy , Inflammation/metabolism , Lipopolysaccharides/pharmacology , MAP Kinase Kinase Kinase 5/drug effects , Macrophages/metabolism , Mice, Inbred C57BL , Mice, Knockout , Neutrophil Infiltration/drug effects , Signal Transduction/drug effectsABSTRACT
Hypersensitivity pneumonitis (HP) is an immune-mediated interstitial lung disease that develops following repeated exposure to inhaled environmental antigens. The disease results in alveolitis and granuloma formation and may progress to a chronic form associated with fibrosis; a greater understanding of the immunopathogenic mechanisms leading to chronic HP is needed. We used the Saccharopolyspora rectivirgula (SR) mouse model of HP to determine the extent to which a switch to a Th2-type immune response is associated with chronic HP. Exposure of wild-type (WT) and tlr2/9(-/-) mice to SR for 14 wk resulted in neutrophilic and lymphocytic alveolitis that was not dependent on Toll-like receptors (TLRs) 2 and 9. Long-term exposure of WT mice to SR resulted in a significant increase in collagen deposition, protein leakage, and IL-1α accompanied by a decrease in quasistatic compliance and total lung capacity compared with unexposed mice. This was associated with an increase in IL-17 but not IL-4 production or recruitment of Th2 cells. tlr2/9(-/-) mice exhibited an increase in protein leakage but less IL-1α and collagen deposition in the lungs compared with WT mice, yet they still displayed a decrease in quasistatic compliance, although total lung capacity was not affected. These mice exhibited an increase in both IL-13 and IL-17, which suggests that IL-13 may ameliorate some of the lung damage caused by long-term SR exposure. Our results suggest that lung pathology following long-term SR exposure in WT mice is associated with the IL-17 response and that TLRs 2 and 9 may inhibit the development of the IL-13/Th2 response.
Subject(s)
Alveolitis, Extrinsic Allergic/immunology , Alveolitis, Extrinsic Allergic/pathology , Saccharopolyspora , Th2 Cells/cytology , Animals , Cytokines/biosynthesis , Female , Interleukin-17/immunology , Mice, Inbred C57BL , Mice, Knockout , Th2 Cells/immunology , Toll-Like Receptor 2/deficiency , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 9/deficiency , Toll-Like Receptor 9/metabolismABSTRACT
Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 T cell responses to this fusion in FLC patients and evaluate fusion-specific T cell receptors (TCRs) for use in cellular immunotherapies. We observe that fusion-specific CD8 T cells are rare and that FLC patient TCR repertoires lack large clusters of related TCR sequences characteristic of potent antigen-specific responses, potentially explaining why endogenous immune responses are insufficient to clear FLC tumors. Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity in vivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC.
Subject(s)
Carcinoma, Hepatocellular , Humans , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/therapy , Carcinoma, Hepatocellular/pathology , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/pathology , Cell- and Tissue-Based Therapy , HSP40 Heat-Shock Proteins/genetics , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/geneticsABSTRACT
Multiple Sclerosis (MS) is a chronic neurodegenerative disease with limited therapeutic options. Recombinant Fc multimers (rFc), designed to mirror many of the anti-inflammatory activities of Intravenous Immunoglobulin (IVIG), have been shown to effectively treat numerous immune-mediated diseases in rodents. In this study we used the experimental autoimmune encephalomyelitis (EAE) murine model of MS to test the efficacy of a rFc, M019, that consists of multimers of the Fc portion of IgG2, in inhibiting disease severity. We show that M019 effectively reduced clinical symptoms when given either pre- or post-symptom onset compared to vehicle treated EAE induced mice. M019 was effective in reducing symptoms in both SJL model of relapsing remitting MS as well as the B6 model of chronic disease. M019 binds to FcγR bearing-monocytes both in vivo and in vitro and prevented immune cell infiltration into the CNS of treated mice. The lack of T cell infiltration into the spinal cord was not due to a decrease in T cell priming; there was an equivalent frequency of Th17 cells in the spleens of M019 and vehicle treated EAE induced mice. Surprisingly, there was an increase in chemokines in the sera but not in the CNS of M019 treated mice compared to vehicle treated animals. We postulate that M019 interacts with a FcγR rich monocyte intermediary to prevent T cell migration into the CNS and demyelination.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis , Neurodegenerative Diseases , Animals , Mice , Multiple Sclerosis/drug therapy , Disease Models, Animal , Receptors, IgGABSTRACT
The emergence and availability of closely related clinical isolates of SARS-CoV-2 offers a unique opportunity to identify novel nonsynonymous mutations that may impact phenotype. Global sequencing efforts show that SARS-CoV-2 variants have emerged and then been replaced since the beginning of the pandemic, yet we have limited information regarding the breadth of variant-specific host responses. Using primary cell cultures and the K18-hACE2 mouse, we investigated the replication, innate immune response, and pathology of closely related, clinical variants circulating during the first wave of the pandemic. Mathematical modeling of the lung viral replication of four clinical isolates showed a dichotomy between two B.1. isolates with significantly faster and slower infected cell clearance rates, respectively. While isolates induced several common immune host responses to infection, one B.1 isolate was unique in the promotion of eosinophil-associated proteins IL-5 and CCL11. Moreover, its mortality rate was significantly slower. Lung microscopic histopathology suggested further phenotypic divergence among the five isolates showing three distinct sets of phenotypes: (i) consolidation, alveolar hemorrhage, and inflammation, (ii) interstitial inflammation/septal thickening and peribronchiolar/perivascular lymphoid cells, and (iii) consolidation, alveolar involvement, and endothelial hypertrophy/margination. Together these findings show divergence in the phenotypic outcomes of these clinical isolates and reveal the potential importance of nonsynonymous mutations in nsp2 and ORF8.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Mice , SARS-CoV-2/genetics , Genotype , Phenotype , Inflammation , Mice, Transgenic , Disease Models, Animal , LungABSTRACT
Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 µM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg-1 per day fully protected against a 10× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 EEEV FL93-939-infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg-1 per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD50 VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg-1 per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.
Subject(s)
Encephalitis Virus, Venezuelan Equine , Encephalomyelitis, Eastern Equine , Humans , Horses , Animals , Mice , United States , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Mice, Inbred C57BL , BrainABSTRACT
Hypersensitivity pneumonitis is an interstitial lung disease that results from repeated pulmonary exposure to various organic Ags, including Saccharopolyspora rectivirgula, the causative agent of farmer's lung disease. Although the contributions of proinflammatory mediators to the disease pathogenesis are relatively well documented, the mechanism(s) involved in the initiation of proinflammatory responses against the causative microorganisms and the contribution of signaling molecules involved in the host immune defense have not been fully elucidated. In the current study, we found that S. rectivirgula induces the activation of protein kinase D (PKD)1 in lung cells in vitro and in vivo. Activation of PKD1 by S. rectivirgula was dependent on MyD88. Inhibition of PKD by pharmacological PKD inhibitor Gö6976 and silencing of PKD1 expression by small interfering RNA revealed that PKD1 is indispensable for S. rectivirgula-mediated activation of MAPKs and NF-kappaB and the expression of various proinflammatory cytokines and chemokines. In addition, compared with controls, mice pretreated with Gö6976 showed significantly suppressed alveolitis and neutrophil influx in bronchial alveolar lavage fluid and interstitial lung tissue, as well as substantially decreased myeloperoxidase activity in the lung after pulmonary exposure to S. rectivirgula. These results demonstrate that PKD1 is essential for S. rectivirgula-mediated proinflammatory immune responses and neutrophil influx in the lung. Our findings also imply the possibility that PKD1 is one of the critical factors that play a regulatory role in the development of hypersensitivity pneumonitis caused by microbial Ags and that inhibition of PKD1 activation could be an effective way to control microbial Ag-induced hypersensitivity pneumonitis.
Subject(s)
Antigens, Bacterial/physiology , Farmer's Lung/immunology , Farmer's Lung/pathology , Inflammation Mediators/physiology , Protein Kinase C/physiology , Saccharopolyspora/enzymology , Saccharopolyspora/immunology , Animals , Enzyme Activation/immunology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/biosynthesis , Farmer's Lung/enzymology , Farmer's Lung/microbiology , Inflammation Mediators/metabolism , Lung/enzymology , Lung/immunology , Lung/microbiology , Lung/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/deficiency , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/physiology , NF-kappa B/antagonists & inhibitors , NF-kappa B/biosynthesis , Neutrophil Infiltration/immunology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolismABSTRACT
Protein kinase D1 (PKD1), a ubiquitously expressed serine/threonine kinase, regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, vesicle trafficking, Ag receptor signaling, and pattern recognition receptor signaling. We found previously that exposure to hypersensitivity pneumonitis (HP) inciting Ag Saccharopolyspora rectivirgula leads to the activation of PKD1 in a MyD88-dependent manner in various types of murine cells in vitro and in the mouse lung in vivo. However, it is currently unknown whether PKD1 plays a role in the S. rectivirgula-induced HP. In this study, we investigated contributions of PKD1 on the S. rectivirgula-induced HP using conditional PKD1-insufficient mice. Compared to control PKD1-sufficient mice, PKD1-insufficient mice showed substantially suppressed activation of MAPKs and NF-κB, expression of cytokines and chemokines, and neutrophilic alveolitis after single intranasal exposure to S. rectivirgula The significantly reduced levels of alveolitis, MHC class II surface expression on neutrophils and macrophages, and IL-17A and CXCL9 expression in lung tissue were observed in the PKD1-insufficient mice repeatedly exposed to S. rectivirgula for 5 wk. PKD1-insuficient mice exposed to S. rectivirgula for 5 wk also showed reduced granuloma formation. Our results demonstrate that PKD1 plays an essential role in the initial proinflammatory responses and neutrophil influx in the lung after exposure to S. rectivirgula and substantially contribute to the development of HP caused by repeated exposure to S. rectivirgula Our findings suggest that PKD1 can be an attractive new molecular target for therapy of S. rectivirgula-induced HP.
Subject(s)
Alveolitis, Extrinsic Allergic , Pneumonia , Protein Kinase C/metabolism , Alveolitis, Extrinsic Allergic/metabolism , Animals , Mice , Mice, Inbred C57BL , Pneumonia/chemically induced , Protein Kinases , SaccharopolysporaABSTRACT
COVID-19 patients with severe symptoms still lack antiviral treatment options. Although remdesivir is the only FDA-approved drug for those patients, its efficacy is limited by premature hydrolysis to nucleoside (NUC), low accumulation in the disease-targeted tissue (lungs), and low antiviral potency. In this study, we synthesized a new series of remdesivir analogues by modifying the ProTide moiety. In comparison with remdesivir, the lead compound MMT5-14 showed 2- to 7-fold higher antiviral activity in four variants of SARS-CoV-2. By reducing premature hydrolysis in hamsters, MMT5-14 increased the prodrug concentration by 200- to 300-fold in the plasma and lungs but also enhanced lung accumulation of the active metabolite triphosphate nucleosides (NTP) by 5-fold. Compared to remdesivir, MMT5-14 also increased the intracellular uptake and activation in lung epithelial cells by 4- to 25-fold. These data suggest that MMT5-14 could be a potential antiviral drug to treat COVID-19 patients with severe symptoms.
Subject(s)
COVID-19 Drug Treatment , Prodrugs , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Humans , Lung , Nucleosides , Prodrugs/pharmacology , Prodrugs/therapeutic use , SARS-CoV-2ABSTRACT
Background: The continued emergence of SARS-CoV-2 variants has caused concern that a constantly evolving virus will escape vaccines and antibody therapies. New approaches are needed. Methods: We created and manufactured an ACE2 extracellular domain (ECD) fragment Fc fusion drug candidate, G921, and engineered the compound for enhanced delivery of drug to peripheral tissues by minimizing the size of the ACE2 ECD and by incorporating an Fc domain to enhance transcytosis. G921 was assessed for binding, neutralization, in vivo anti-inflammatory effect, and pharmacokinetic profile. Results: G921 was expressed as an IgG4 Fc fusion protein presenting two ACE2 domains to ACE2 ligands while avoiding risk of infection via antibody-dependent enhancement. G921 strongly binds to the SARS-CoV-2 Wuhan-Hu-1 spike protein and demonstrates further diminished off rate to the spike protein from each of the currently identified variants of concern. G921 demonstrates ACE2 enzymatic activity comparable to positive control and binding to the neonatal Fc receptor (FcRn) without binding to low affinity Fc-gamma receptors (FcγRs). G921 is effective in a concentration-dependent manner in a focus reduction neutralization assay with EC50=16.3±4.2 µg/mL without cytotoxicity in Vero E6 cells when tested at 200 µg/mL in an MTS cell proliferation assay. G921 demonstrates statistically significant reduction of lung inflammation in relevant models of both SARS-CoV-2 and influenza. The pharmacokinetic profile demonstrated dose-dependent exposure with a multi-day half-life in monkeys and rats. Conclusion: G921 data are consistent with both antiviral and anti-inflammatory modes of action. G921 is a novel approach for the prevention and treatment of COVID-19 and possible other diseases characterized by deficiency of ACE2.
ABSTRACT
BACKGROUND: A number of studies have revealed that Francisella tularensis (FT) suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen. RESULTS: Here we describe the characterization of a galU mutant strain of FT live vaccine strain (LVS). We show that the galU mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT) strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the galU mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the galU mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1ß following infection either in vitro or in vivo, indicating that attenuation of the galU mutant strain may be due (in part) to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the galU mutant strain elicits protective immunity to subsequent challenge with WT FT. CONCLUSIONS: Disruption of the galU gene of FTLVS has little (if any) effect on in vivo infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in an accelerated fashion. These results have identified two potentially important virulence mechanisms used by FT. These findings could also have implications for design of a live attenuated vaccine strain of FT because sublethal infection of mice with the galU mutant strain of FTLVS promoted development of protective immunity to WT FTLVS.
Subject(s)
Francisella tularensis/genetics , Francisella tularensis/pathogenicity , Tularemia/microbiology , Tularemia/pathology , UTP-Glucose-1-Phosphate Uridylyltransferase/deficiency , Virulence Factors/deficiency , Animals , Chemokines/metabolism , Disease Models, Animal , Francisella tularensis/immunology , Humans , Interleukin-1beta/immunology , Lung/immunology , Lung/microbiology , Mice , Mice, Inbred C57BL , Neutrophils/immunology , Pneumonia, Bacterial/microbiology , Pneumonia, Bacterial/pathology , Rodent Diseases/microbiology , Rodent Diseases/pathology , Toll-Like Receptor 2/immunology , Toll-Like Receptor 4/immunology , VirulenceABSTRACT
The success of Intravenous Immunoglobulin in treating autoimmune and inflammatory processes such as immune thrombocytopenia purpura and Kawasaki disease has led to renewed interest in developing recombinant molecules capable of recapitulating these therapeutic effects. The anti-inflammatory properties of IVIG are, in part, due to the Fc region of the IgG molecule, which interacts with activating or inhibitory Fcγ receptors (FcγRs), the neonatal Fc Receptor, non-canonical FcRs expressed by immune cells and complement proteins. In most cases, Fc interactions with these cognate receptors are dependent upon avidity-avidity which naturally occurs when polyclonal antibodies recognize unique antigens on a given target. The functional consequences of these avid interactions include antibody dependent cell-mediated cytotoxicity, antibody dependent cell phagocytosis, degranulation, direct killing, and/or complement activation-all of which are associated with long-term immunomodulatory effects. Many of these immunologic effects can be recapitulated using recombinant or non-recombinant approaches to induce Fc multimerization, affording the potential to develop a new class of therapeutics. In this review, we discuss the history of tolerance induction by immune complexes that has led to the therapeutic development of artificial Fc bearing immune aggregates and recombinant Fc multimers. The contribution of structure, aggregation and N-glycosylation to human IgG: FcγR interactions and the functional effect(s) of these interactions are reviewed. Understanding the mechanisms by which Fc multimers induce tolerance and attempts to engineer Fc multimers to target specific FcγRs and/or specific effector functions in autoimmune disorders is explored in detail.
Subject(s)
Autoimmune Diseases/therapy , Biological Therapy/methods , Immunoglobulin Fc Fragments/genetics , Animals , Complement Activation , Genetic Engineering , Humans , Immunoglobulins, Intravenous/pharmacology , Protein Multimerization , Receptors, IgG/metabolismABSTRACT
Pneumonia is the leading cause of infectious related death costing 12 billion dollars annually in the United States alone. Despite improvements in clinical care, total mortality remains around 4%, with inpatient mortality reaching 5-10%. For unknown reasons, mortality risk remains high even after hospital discharge and there is a need to identify those patients most at risk. Also of importance, clinical symptoms alone do not distinguish viral from bacterial infection which may delay appropriate treatment and may contribute to short-term and long-term mortality. Biomarkers have the potential to provide point of care diagnosis, identify high-risk patients, and increase our understanding of the biology of disease. However, there have been mixed results on the diagnostic performance of many of the analytes tested to date. Urine represents a largely untapped source for biomarker discovery and is highly accessible. To test this hypothesis, we collected urine from hospitalized patients with community-acquired pneumonia (CAP) and performed a comprehensive screen for urinary tract microbiota signatures, metabolite, and cytokine profiles. CAP patients were diagnosed with influenza or bacterial (Streptococcus pneumoniae and Staphylococcus aureus) etiologies and compared with healthy volunteers. Microbiome signatures showed marked shifts in taxonomic levels in patients with bacterial etiology versus influenza and CAP versus normal. Predictive modeling of 291 microbial and metabolite values achieved a + 90% accuracy with LASSO in predicting specific pneumonia etiology. This study demonstrates that urine from patients hospitalized with pneumonia may serve as a reliable and accessible sample to evaluate biomarkers that may diagnose etiology and predict clinical outcomes.
Subject(s)
Community-Acquired Infections/microbiology , Community-Acquired Infections/virology , Cytokines/urine , Inpatients/statistics & numerical data , Urine/microbiology , Adult , Biomarkers/urine , Community-Acquired Infections/diagnosis , Community-Acquired Infections/mortality , Female , Hospital Mortality , Humans , Male , Middle Aged , Predictive Value of Tests , Prognosis , Risk , United StatesABSTRACT
Hypersensitivity pneumonitis is an interstitial lung disease that is characterized by alveolitis, granuloma formation, and in some patients, fibrosis. Using the Saccharopolyspora rectivirgula animal model of Farmer's lung disease, our laboratory has demonstrated that neutrophils play a critical role in IFN-gamma production during the acute phase of the disease. As IFN-gamma is necessary for granuloma formation, it is important to identify the factors that lead to neutrophil recruitment during disease. To begin to identify the pattern recognition receptors (PRRs) that initiate chemokine production, leading to neutrophil recruitment following S. rectivirgula exposure, we examined the role of MyD88 and TLR2. Our results demonstrate that neutrophil recruitment, as measured by flow cytometry and the myeloperoxidase assay, was abolished in the absence of MyD88 following S. rectivirgula exposure. The decrease in neutrophil recruitment was likely a result of a significant decrease in production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine. These results suggest that S. rectivirgula interacts with PRRs that are upstream of the MyD88 pathway to initiate cytokine and chemokine production. In vitro studies suggest that S. rectivirgula can interact with TLR2, and stimulation of adherent cells from TLR2 knockout (KO) mice with S. rectivirgula resulted in a significant decrease in MIP-2 production. However, TLR2 KO mice did not have a reduction in neutrophil recruitment compared with wild-type mice following S. rectivirgula exposure. The results from our studies suggest that one or more PRR(s) upstream of MyD88 are necessary for neutrophil recruitment following S. rectivirgula exposure.
Subject(s)
Alveolitis, Extrinsic Allergic/physiopathology , Gram-Negative Bacterial Infections/physiopathology , Myeloid Differentiation Factor 88/physiology , Neutrophils/physiology , Alveolitis, Extrinsic Allergic/microbiology , Animals , Bronchoalveolar Lavage , Bronchoalveolar Lavage Fluid/cytology , Crosses, Genetic , Female , HeLa Cells , Humans , Lung/pathology , Lung/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Saccharopolyspora , Toll-Like Receptor 2/deficiency , Toll-Like Receptor 2/physiologyABSTRACT
Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5â¯mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48â¯h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25â¯mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase.
Subject(s)
Benzamides , Benzamidines , Drug Resistance, Viral/genetics , Encephalitis Virus, Eastern Equine/drug effects , Encephalitis Virus, Venezuelan Equine/drug effects , Piperazines , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Benzamides/chemical synthesis , Benzamides/pharmacology , Benzamidines/chemical synthesis , Benzamidines/pharmacology , Cell Line , Encephalomyelitis, Eastern Equine/drug therapy , Encephalomyelitis, Eastern Equine/prevention & control , Encephalomyelitis, Venezuelan Equine/drug therapy , Encephalomyelitis, Venezuelan Equine/prevention & control , Genes, Viral , Mice , Mutation , Piperazines/chemical synthesis , Piperazines/pharmacologyABSTRACT
Fibroblast growth factor-23 (FGF-23) is a bone-derived hormone that activates FGFR/α-Klotho binary complexes in the kidney renal tubules to regulate phosphate reabsorption and vitamin D metabolism. The objective of this review is to discuss the emerging data that show that FGF-23 has functions beyond regulation of mineral metabolism, including roles in innate immune and hemodynamic responses. Excess FGF-23 is associated with inflammation and adverse infectious outcomes, as well as increased morbidity and mortality, particularly in patients with chronic kidney disease. Enhancer elements in the FGF-23 promoter have been identified that mediate the effects of inflammatory cytokines to stimulate FGF-23 gene transcription in bone. In addition, inflammation induces ectopic expression of FGF-23 and α-Klotho in macrophages that do not normally express FGF-23 or its binary receptor complexes. These observations suggest that FGF-23 may play an important role in regulating innate immunity through multiple potential mechanisms. Circulating FGF-23 acts as a counter-regulatory hormone to suppress 1,25D production in the proximal tubule of the kidney. Since vitamin D deficiency may predispose infectious and cardiovascular diseases, FGF-23 effects on innate immune responses may be due to suppression of 1,25D production. Alternatively, systemic and locally produced FGF-23 may modulate immune functions through direct interactions with myeloid cells, including macrophages and polymorphonuclear leukocytes to impair immune cell functions. Short-acting small molecules that reversibly inhibit FGF-23 offer the potential to block pro-inflammatory and cardiotoxic effects of FGF-23 with less side effects compared with FGF-23 blocking antibodies that have the potential to cause hyperphosphatemia and soft tissue calcifications in animal models. In conclusion, there are several mechanisms by which FGF-23 impacts the innate immune system and further investigation is critical for the development of therapies to treat diseases associated with elevated FGF-23.