Biology subjects

Gorbunova, E.

Publications and source records attributed to Gorbunova, E..

4 recordsLinked to original sources

bioRxiv2023

Age-dependent Powassan Virus Lethality is Directed by Glial Cell Activation and Divergent Neuroinflammatory Cytokine Responses in a Murine Model

Powassan virus (POWV) is an emergent tick-borne flavivirus that causes fatal encephalitis in the elderly and long-term neurologic sequelae in survivors. How age contributes to severe POWV encephalitis remains an enigma and there are currently no animal models that reflect age-dependent POWV neuropathology. Inoculating C57BL/6 mice with a POWV strain (LI9) currently circulating in Ixodes ticks, resulted in age-dependent POWV lethality with overt spongiform brain damage 10-15 dpi. Infection of 50 week old mice resulted in 82% lethality 10-15 dpi that was sequentially reduced by age to 7.1% in 10 week old mice. LI9 encephalitis resulted in early neuronal depletion, with severe CNS damage, persistent inflammatory gliosis and long-term spongiform pathology in survivors (30 dpi). In all mice POWV LI9 was neuroinvasive and reached maximum POWV loads in the CNS 10 dpi. Coincident with murine lethality, in 50 week old mice maximum POWV CNS levels persisted 15 dpi, while instead decreasing by 2-4 logs in 10-30 week old mice. Although glial cells were highly activated in all POWV infected mice, differences in age-dependent CNS cytokine responses were striking 15 dpi. In 50 week old mice POWV induced Th1-type cytokines (IFN{gamma}, IL-2, IL-12, IL-4, TNF, IL-6), suggesting a pro-inflammatory M1 microglial activation cascade. In contrast, POWV induced Th2-type cytokines (IL-10, TGF{beta}, IL-4) in 10 week old mice consistent with a neuroprotective M2 microglial phenotype. These findings reflect differences in neurodegenerative versus neuroprotective glial cell responses that correlate with divergent CNS viral clearance and age-dependent POWV LI9 lethality. Discrete age-dependent CNS cytokine responses suggest neuroinflammatory targets as potential POWV therapeutics. These studies establish a highly lethal POWV murine model and reveal a hyperinflammatory mechanism of age-dependent POWV lethality that mirrors human POWV severity and long-term CNS sequelae in the elderly. ImportancePowassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. Findings demonstrate that POWV load and discrete glial cell cytokine responses in the CNS are critical determinants of age-dependent POWV lethality. POWV age-independently activates microglia and astrocytes, but directs neuroprotective Th2 cytokine responses in 10 week old mice and distinct pro-inflammatory Th1 cytokine responses in the CNS of 50 week old mice. This reveals roles for a hyperinflammatory CNS cytokine cascade in age-dependent POWV lethality, and protective anti-inflammatory cytokines in murine survival. Notably, results define potential therapeutic targets and rationalize approaches for preventing severe POWV encephalitis that may be broadly applicable to neurodegenerative diseases. This age-dependent murine POWV model permits analysis of vaccines, and therapeutics that prevent POWV neuroinvasion or resolve severe POWV encephalitis in the elderly.

microbiology↗

bioRxiv2023

Age-dependent Powassan Virus Lethality and Neuropathogenesis in Mice

Powassan viruses (POWV) are emergent tick-borne flaviviruses that cause severe neurologic disease in humans. Subcutaneous inoculation of C57BL/6 mice with POWV (strain LI9) resulted in overt brain damage resembling spongiform encephalitis. Noting higher POWV lethality in older mice, we assessed neurovirulence as a function of age. We found that POWV LI9 inoculation was lethal in 80% of 50 wk old mice, 10-15 dpi, and that lethality was sequentially reduced in 40, 30, 20, 10 wk old mice to <10%. Lethality was conferred by 2-20 POWV FFUs, and POWV neuropathology was evident as early as 5 dpi, with lethal disease 10-15 dpi correlated with sustained POWV RNA levels in brains of aged mice. Histology of POWV infected 50 wk old murine brains revealed severe spongiform neuronal necrosis, microgliosis, and inflammation with increased brainstem and cerebellar damage. These findings delineate an age-dependent murine model of lethal POWV infection that mirrors human POWV disease and permits analysis of age-dependent neurovirulence determinants. SignificanceOur findings establish a novel age-dependent lethal animal model to study encephalitic POWV disease in vivo. These initial findings demonstrate that following peripheral inoculation, non-neuroadapted POWV LI9 is neuroinvasive and enters the brains of young and aged mice. However, POWV LI9 lethality is strictly age-dependent and correlated with increased viral load in the brains of aged mice. POWV rapidly directs neuronal loss and spongiform lesions, microglial activation and causes prolonged inflammation that fails to clear POWV from the brains of aged mice. Our results provide a lethal murine model of POWV neurovirulence that mirrors the prevalence of severe human POWV encephalitis in the elderly. This lethal murine POWV model provides mechanisms for defining POWV protective responses of the young, revealing determinants of age-dependent POWV lethality and evaluating potential POWV therapeutics. SUMMARYPowassan virus is an emerging tick-borne flavivirus linked to severe neurologic disease in aged individuals. Here we describe an age-dependent mouse model of POWV pathogenesis. SUBJECTSPowassan virus, flavivirus, neurovirulence, neuroinvasion, neurotropic, spongiform encephalopathy, microgliosis, neuroinflammation

microbiology↗

bioRxiv2022

Genetic and environmental circadian disruption induce metabolic impairment through changes in the gut microbiome

ObjectiveInternal clocks time behavior and physiology, including the gut microbiome in a circadian ([~]24 h) manner. Mismatch between internal and external time, e.g. during shift work, disrupts circadian system coordination promoting the development of obesity and type 2 diabetes (T2D). Conversely, body weight changes induce microbiota dysbiosis. The relationship between circadian disruption and microbiota dysbiosis in metabolic diseases, however, remains largely unknown. MethodsCore and accessory clock gene expression in different gastrointestinal (GI) tissues were determined by qPCR in two different models of circadian disruption - mice with Bmal1 deficiency in the circadian pacemaker, the suprachiasmatic nucleus (Bmal1SCNfl/-), and wild-type mice exposed to simulated shift work (SSW). Body composition and energy balance were evaluated by nuclear magnetic resonance (NMR), bomb calorimetry, food intake and running-wheel activity. Intestinal permeability was measured in an Ussing chamber. Microbiota composition and functionality were evaluated by 16S rRNA gene amplicon sequencing, PICRUST2.0 analysis and targeted metabolomics. Finally, microbiota transfer was conducted to evaluate the functional impact of SSW-associated microbiota on the hosts physiology. ResultsBoth chronodisruption models show desynchronization within and between peripheral clocks in GI tissues and reduced microbial rhythmicity, in particular in taxa involved in short-chain fatty acid (SCFA) fermentation and lipid metabolism. In Bmal1SCNfl/- mice, loss of rhythmicity in microbial functioning associates with previously shown increased body weight, dysfunctional glucose homeostasis and adiposity. Similarly, we observe an increase in body weight in SSW mice. Germ-free colonization experiments with SSW- associated microbiota mechanistically link body weight gain to microbial changes. Moreover, alterations in expression of peripheral clock genes as well as clock-controlled genes (CCGs) relevant for metabolic functioning of the host were observed in recipients, indicating a bidirectional relationship between microbiota rhythmicity and peripheral clock regulation. ConclusionsCollectively, our data suggest that loss of rhythmicity in bacteria taxa and their products, which likely originates in desynchronization of intestinal clocks, promotes metabolic abnormalities during shift work.

physiology↗

bioRxiv2020

Recombinant ACE2 Expression is Required for SARS-CoV-2 to Infect Primary Human Endothelial Cells and Induce Inflammatory and Procoagulative Responses

SARS-CoV-2 causes COVID-19, an acute respiratory distress syndrome (ARDS) characterized by pulmonary edema, viral pneumonia, multiorgan dysfunction, coagulopathy and inflammation. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) receptors to infect and damage ciliated epithelial cells in the upper respiratory tract. In alveoli, gas exchange occurs across an epithelial-endothelial barrier that ties respiration to endothelial cell (EC) regulation of edema, coagulation and inflammation. How SARS-CoV-2 dysregulates vascular functions to cause ARDS in COVID-19 patients remains an enigma focused on dysregulated EC responses. Whether SARS-CoV-2 directly or indirectly affects functions of the endothelium remains to be resolved and critical to understanding SARS-CoV-2 pathogenesis and therapeutic targets. We demonstrate that primary human ECs lack ACE2 receptors at protein and RNA levels, and that SARS-CoV-2 is incapable of directly infecting ECs derived from pulmonary, cardiac, brain, umbilical vein or kidney tissues. In contrast, pulmonary ECs transduced with recombinant ACE2 receptors are infected by SARS-CoV-2 and result in high viral titers ([~]1x107/ml), multinucleate syncytia and EC lysis. SARS-CoV-2 infection of ACE2-expressing ECs elicits procoagulative and inflammatory responses observed in COVID-19 patients. The inability of SARS-CoV-2 to directly infect and lyse ECs without ACE2 expression explains the lack of vascular hemorrhage in COVID-19 patients and indicates that the endothelium is not a primary target of SARS-CoV-2 infection. These findings are consistent with SARS-CoV-2 indirectly activating EC programs that regulate thrombosis and endotheliitis in COVID-19 patients, and focus strategies on therapeutically targeting epithelial and inflammatory responses that activate the endothelium or initiate limited ACE2 independent EC infection. ImportanceSARS-CoV-2 infects pulmonary epithelial cells through ACE2 receptors and causes ARDS. COVID-19 causes progressive respiratory failure resulting from diffuse alveolar damage and systemic coagulopathy, thrombosis and capillary inflammation that tie alveolar responses to EC dysfunction. This has prompted theories that SARS-CoV-2 directly infects ECs through ACE2 receptors, yet SARS-CoV-2 antigen has not been co-localized with ECs and prior studies indicate that ACE2 co-localizes with alveolar epithelial cells and vascular smooth muscle cells, not ECs. Here we demonstrate that primary human ECs derived from lung, kidney, heart, brain and umbilical veins require expression of recombinant ACE2 receptors in order to be infected by SARS-CoV-2. However, SARS-CoV-2 lytically infects ACE2-ECs and elicits procoagulative and inflammatory responses observed in COVID-19 patients. These findings suggest a novel mechanism of COVID-19 pathogenesis resulting from indirect EC activation, or infection of a small subset of ECs by an ACE2 independent mechanism, that transform rationales and targets for therapeutic intervention.

microbiology↗