Hypothalamic hormone deficiency enables physiological anorexia.

Mammalian hibernators survive prolonged periods of cold and resource scarcity by temporarily modulating normal physiological functions, but the mechanisms underlying these adaptations are poorly understood. The hibernation cycle of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) lasts for 5-7 months and comprises weeks of hypometabolic, hypothermic torpor interspersed with 24-48-hour periods of an active-like interbout arousal (IBA) state. We show that ground squirrels, who endure the entire hibernation season without food, have negligible hunger during IBAs. These squirrels exhibit reversible inhibition of the hypothalamic feeding center, such that hypothalamic arcuate nucleus neurons exhibit reduced sensitivity to the orexigenic and anorexigenic effects of ghrelin and leptin, respectively. However, hypothalamic infusion of thyroid hormone during an IBA is sufficient to rescue hibernation anorexia. Our results reveal that thyroid hormone deficiency underlies hibernation anorexia and demonstrate the functional flexibility of the hypothalamic feeding center.

A multiomics analysis of direct interkingdom dynamics between influenza A virus and Streptococcus pneumoniae uncovers host-independent changes to bacterial virulence fitness.

BACKGROUND: For almost a century, it has been recognized that influenza A virus (IAV) infection can promote the development of secondary bacterial infections (SBI) mainly caused by Streptococcus pneumoniae (Spn). Recent observations have shown that IAV is able to directly bind to the surface of Spn. To gain a foundational understanding of how direct IAV-Spn interaction alters bacterial biological fitness we employed combinatorial multiomic and molecular approaches. RESULTS: Here we show IAV significantly remodels the global transcriptome, proteome and phosphoproteome profiles of Spn independently of host effectors. We identified Spn surface proteins that interact with IAV proteins (hemagglutinin, nucleoprotein, and neuraminidase). In addition, IAV was found to directly modulate expression of Spn virulence determinants such as pneumococcal surface protein A, pneumolysin, and factors associated with antimicrobial resistance among many others. Metabolic pathways were significantly altered leading to changes in Spn growth rate. IAV was also found to drive Spn capsule shedding and the release of pneumococcal surface proteins. Released proteins were found to be involved in evasion of innate immune responses and actively reduced human complement hemolytic and opsonizing activity. IAV also led to phosphorylation changes in Spn proteins associated with metabolism and bacterial virulence. Validation of proteomic data showed significant changes in Spn galactose and glucose metabolism. Furthermore, supplementation with galactose rescued bacterial growth and promoted bacterial invasion, while glucose supplementation led to enhanced pneumolysin production and lung cell apoptosis. CONCLUSIONS: Here we demonstrate that IAV can directly modulate Spn biology without the requirement of host effectors and support the notion that inter-kingdom interactions between human viruses and commensal pathobionts can promote bacterial pathogenesis and microbiome dysbiosis.

Pandemic Influenza Infection Promotes Streptococcus pneumoniae Infiltration, Necrotic Damage, and Proteomic Remodeling in the Heart.

For over a century, it has been reported that primary influenza infection promotes the development of a lethal form of bacterial pulmonary disease. More recently, pneumonia events caused by both viruses and bacteria have been directly associated with cardiac damage. Importantly, it is not known whether viral-bacterial synergy extends to extrapulmonary organs such as the heart. Using label-free quantitative proteomics and molecular approaches, we report that primary infection with pandemic influenza A virus leads to increased Streptococcus pneumoniae translocation to the myocardium, leading to general biological alterations. We also observed that each infection alone led to proteomic changes in the heart, and these were exacerbated in the secondary bacterial infection (SBI) model. Gene ontology analysis of significantly upregulated proteins showed increased innate immune activity, oxidative processes, and changes to ion homeostasis during SBI. Immunoblots confirmed increased complement and antioxidant activity in addition to increased expression of angiotensin-converting enzyme 2. Using an in vitro model of sequential infection in human cardiomyocytes, we observed that influenza enhances S. pneumoniae cytotoxicity by promoting oxidative stress enhancing bacterial toxin-induced necrotic cell death. Influenza infection was found to increase receptors that promote bacterial adhesion, such as polymeric immunoglobulin receptor and fibronectin leucine-rich transmembrane protein 1 in cardiomyocytes. Finally, mice deficient in programmed necrosis (i.e., necroptosis) showed enhanced innate immune responses, decreased virus-associated pathways, and promotion of mitochondrial function upon SBI. The presented results provide the first in vivo evidence that influenza infection promotes S. pneumoniae infiltration, necrotic damage, and proteomic remodeling of the heart. IMPORTANCE Adverse cardiac events are a common complication of viral and bacterial pneumonia. For over a century, it has been recognized that influenza infection promotes severe forms of pulmonary disease mainly caused by the bacterium Streptococcus pneumoniae. The extrapulmonary effects of secondary bacterial infections to influenza virus are not known. In the present study, we used a combination of quantitative proteomics and molecular approaches to assess the underlying mechanisms of how influenza infection promotes bacteria-driven cardiac damage and proteome remodeling. We further observed that programmed necrosis (i.e., necroptosis) inhibition leads to reduced damage and proteome changes associated with health.

Kinetic Multi-omic Analysis of Responses to SARS-CoV-2 Infection in a Model of Severe COVID-19.

The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is poorly understood due to a lack of an animal model that recapitulates severe human disease. Here, we report a Syrian hamster model that develops progressive lethal pulmonary disease that closely mimics severe coronavirus disease 2019 (COVID-19). We evaluated host responses using a multi-omic, multiorgan approach to define proteome, phosphoproteome, and transcriptome changes. These data revealed both type I and type II interferon-stimulated gene and protein expression along with a progressive increase in chemokines, monocytes, and neutrophil-associated molecules throughout the course of infection that peaked in the later time points correlating with a rapidly developing diffuse alveolar destruction and pneumonia that persisted in the absence of active viral infection. Extrapulmonary proteome and phosphoproteome remodeling was detected in the heart and kidneys following viral infection. Together, our results provide a kinetic overview of multiorgan host responses to severe SARS-CoV-2 infection in vivo. IMPORTANCE The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has created an urgent need to understand the pathogenesis of this infection. These efforts have been impaired by the lack of animal models that recapitulate severe coronavirus disease 2019 (COVID-19). Here, we report a hamster model that develops severe COVID-19-like disease following infection with human isolates of SARS-CoV-2. To better understand pathogenesis, we evaluated changes in gene transcription and protein expression over the course of infection to provide an integrated multiorgan kinetic analysis of the host response to infection. These data reveal a dynamic innate immune response to infection and corresponding immune pathologies consistent with severe human disease. Altogether, this model will be useful for understanding the pathogenesis of severe COVID-19 and for testing interventions.

Streptococcus pneumoniae binds to host GAPDH on dying lung epithelial cells worsening secondary infection following influenza.

Streptococcus pneumoniae (Spn) alone and during co-infection with influenza A virus (IAV) can result in severe pneumonia with mortality. Pneumococcal surface protein A (PspA) is an established virulence factor required for Spn evasion of lactoferricin and C-reactive protein-activated complement-mediated killing. Herein, we show that PspA functions as an adhesin to dying host cells. We demonstrate that PspA binds to host-derived glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bound to outward-flipped phosphatidylserine residues on dying host cells. PspA-mediated adhesion was to apoptotic, pyroptotic, and necroptotic cells, but not healthy lung cells. Using isogenic mutants of Spn, we show that PspA-GAPDH-mediated binding to lung cells increases pneumococcal localization in the lower airway, and this is enhanced as a result of pneumolysin exposure or co-infection with IAV. PspA-mediated binding to GAPDH requires amino acids 230-281 in its α-helical domain with intratracheal inoculation of this PspA fragment alongside the bacteria reducing disease severity in an IAV/Spn pneumonia model.

Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence.

RATIONALE: Patients with and without cardiovascular diseases have been shown to be at risk of influenza-mediated cardiac complications. Recent clinical reports support the notion of a direct link between laboratory-confirmed influenza virus infections and adverse cardiac events. OBJECTIVE: Define the molecular mechanisms underlying influenza virus-induced cardiac pathogenesis after resolution of pulmonary infection and the role of necroptosis in this process. METHODS AND RESULTS: Hearts from wild-type and necroptosis-deficient (MLKL [mixed lineage kinase domain-like protein]-KO) mice were dissected 12 days after initial influenza A virus (IAV) infection when viral titers were undetectable in the lungs. Immunofluorescence microscopy and plaque assays showed presence of viable IAV particles in the myocardium without generation of interferon responses. Global proteome and phosphoproteome analyses using high-resolution accurate mass-based LC-MS/MS and label-free quantitation showed that the global proteome as well as the phosphoproteome profiles were significantly altered in IAV-infected mouse hearts in a strain-independent manner. Necroptosis-deficient mice had increased survival and reduced weight loss post-IAV infection, as well as increased antioxidant and mitochondrial function, indicating partial protection to IAV infection. These findings were confirmed in vitro by pretreatment of human and rat myocytes with antioxidants or necroptosis inhibitors, which blunted oxidative stress and mitochondrial damage after IAV infection. CONCLUSIONS: This study provides the first evidence that the cardiac proteome and phosphoproteome are significantly altered post-pulmonary influenza infection. Moreover, viral particles can persist in the heart after lung clearance, altering mitochondrial function and promoting cell death without active replication and interferon responses. Finally, our findings show inhibition of necroptosis or prevention of mitochondrial damage as possible therapeutic interventions to reduce cardiac damage during influenza infections. Graphic Abstract: A graphic abstract is available for this article.

Global Proteome and Phosphoproteome Characterization of Sepsis-induced Kidney Injury.

Sepsis-induced acute kidney injury (S-AKI) is the most common complication in hospitalized and critically ill patients, highlighted by a rapid decline of kidney function occurring a few hours or days after sepsis onset. Systemic inflammation elicited by microbial infections is believed to lead to kidney damage under immunocompromised conditions. However, although AKI has been recognized as a disease with long-term sequelae, partly because of the associated higher risk of chronic kidney disease (CKD), the understanding of kidney pathophysiology at the molecular level and the global view of dynamic regulations in situ after S-AKI, including the transition to CKD, remains limited. Existing studies of S-AKI mainly focus on deriving sepsis biomarkers from body fluids. In the present study, we constructed a mid-severity septic murine model using cecal ligation and puncture (CLP), and examined the temporal changes to the kidney proteome and phosphoproteome at day 2 and day 7 after CLP surgery, corresponding to S-AKI and the transition to CKD, respectively, by employing an ultrafast and economical filter-based sample processing method combined with the label-free quantitation approach. Collectively, we identified 2,119 proteins and 2950 phosphosites through multi-proteomics analyses. Among them, we identified an array of highly promising candidate marker proteins indicative of disease onset and progression accompanied by immunoblot validations, and further denoted the pathways that are specifically responsive to S-AKI and its transition to CKD, which include regulation of cell metabolism regulation, oxidative stress, and energy consumption in the diseased kidneys. Our data can serve as an enriched resource for the identification of mechanisms and biomarkers for sepsis-induced kidney diseases.

Influenza-Induced Oxidative Stress Sensitizes Lung Cells to Bacterial-Toxin-Mediated Necroptosis.

Pneumonias caused by influenza A virus (IAV) co- and secondary bacterial infections are characterized by their severity and high mortality rate. Previously, we have shown that bacterial pore-forming toxin (PFT)-mediated necroptosis is a key driver of acute lung injury during bacterial pneumonia. Here, we evaluate the impact of IAV on PFT-induced acute lung injury during co- and secondary Streptococcus pneumoniae (Spn) infection. We observe that IAV synergistically sensitizes lung epithelial cells for PFT-mediated necroptosis in vitro and in murine models of Spn co-infection and secondary infection. Pharmacoelogical induction of oxidative stress without virus sensitizes cells for PFT-mediated necroptosis. Antioxidant treatment or inhibition of necroptosis reduces disease severity during secondary bacterial infection. Our results advance our understanding on the molecular basis of co- and secondary bacterial infection to influenza and identify necroptosis inhibition and antioxidant therapy as potential intervention strategies.

Th17 lymphocytes drive vascular and neuronal deficits in a mouse model of postinfectious autoimmune encephalitis.

Antibodies against neuronal receptors and synaptic proteins are associated with a group of ill-defined central nervous system (CNS) autoimmune diseases termed autoimmune encephalitides (AE), which are characterized by abrupt onset of seizures and/or movement and psychiatric symptoms. Basal ganglia encephalitis (BGE), representing a subset of AE syndromes, is triggered in children by repeated group A Streptococcus (GAS) infections that lead to neuropsychiatric symptoms. We have previously shown that multiple GAS infections of mice induce migration of Th17 lymphocytes from the nose into the brain, causing blood-brain barrier (BBB) breakdown, extravasation of autoantibodies into the CNS, and loss of excitatory synapses within the olfactory bulb (OB). Whether these pathologies induce functional olfactory deficits, and the mechanistic role of Th17 lymphocytes, is unknown. Here, we demonstrate that, whereas loss of excitatory synapses in the OB is transient after multiple GAS infections, functional deficits in odor processing persist. Moreover, mice lacking Th17 lymphocytes have reduced BBB leakage, microglial activation, and antibody infiltration into the CNS, and have their olfactory function partially restored. Th17 lymphocytes are therefore critical for selective CNS entry of autoantibodies, microglial activation, and neural circuit impairment during postinfectious BGE.

Hello from the Other Side: How Autoantibodies Circumvent the Blood-Brain Barrier in Autoimmune Encephalitis.

Antibodies against neuronal receptors and synaptic proteins are associated with autoimmune encephalitides (AE) that produce movement and psychiatric disorders. In order to exert their pathological effects on neural circuits, autoantibodies against central nervous system (CNS) targets must gain access to the brain and spinal cord by crossing the blood-brain barrier (BBB), a tightly regulated gateway formed by endothelial cells lining CNS blood vessels. To date, the pathogenic mechanisms that underlie autoantibody-triggered encephalitic syndromes are poorly understood, and how autoantibodies breach the barrier remains obscure for almost all AE syndromes. The relative importance of cellular versus humoral immune mechanisms for disease pathogenesis also remains largely unexplored. Here, we review the proposed triggers for various autoimmune encephalopathies and their animal models, as well as basic structural features of the BBB and how they differ among various CNS regions, a feature that likely underlies some regional aspects of autoimmune encephalitis pathogenesis. We then discuss the routes that antibodies and immune cells employ to enter the CNS and their implications for AE. Finally, we explore future therapeutic strategies that may either preserve or restore barrier function and thereby limit immune cell and autoantibody infiltration into the CNS. Recent mechanistic insights into CNS autoantibody entry indicate promising future directions for therapeutic intervention beyond current, short-lived therapies that eliminate circulating autoantibodies.

Group A Streptococcus intranasal infection promotes CNS infiltration by streptococcal-specific Th17 cells.

Group A streptococcal (GAS) infection induces the production of Abs that cross-react with host neuronal proteins, and these anti-GAS mimetic Abs are associated with autoimmune diseases of the CNS. However, the mechanisms that allow these Abs to cross the blood-brain barrier (BBB) and induce neuropathology remain unresolved. We have previously shown that GAS infection in mouse models induces a robust Th17 response in nasal-associated lymphoid tissue (NALT). Here, we identified GAS-specific Th17 cells in tonsils of humans naturally exposed to GAS, prompting us to explore whether GAS-specific CD4+ T cells home to mouse brains following i.n. infection. Intranasal challenge of repeatedly GAS-inoculated mice promoted migration of GAS-specific Th17 cells from NALT into the brain, BBB breakdown, serum IgG deposition, microglial activation, and loss of excitatory synaptic proteins under conditions in which no viable bacteria were detected in CNS tissue. CD4+ T cells were predominantly located in the olfactory bulb (OB) and in other brain regions that receive direct input from the OB. Together, these findings provide insight into the immunopathology of neuropsychiatric complications that are associated with GAS infections and suggest that crosstalk between the CNS and cellular immunity may be a general mechanism by which infectious agents exacerbate symptoms associated with other CNS autoimmune disorders.

Position of neocortical neurons transfected at different gestational ages with shRNA targeted against candidate dyslexia susceptibility genes.

Developmental dyslexia is a language learning disorder that affects approximately 4-10% of the population. A number of candidate dyslexia susceptibility genes have been identified, including DCDC2 and KIAA0319 on Chromosome (Chr) 6p22.2 and DYX1C1 on Chr 15q21. Embryonic knockdown of the function of homologs of these genes in rat neocortical projection cell progenitors by in utero electroporation of plasmids encoding small hairpin RNA (shRNA) revealed that all three genes disrupted neuronal migration to the neocortex. Specifically, this disruption would result in heterotopia formation (Dyx1c1 and Kiaa0319) and/or overmigration past their expected laminar location (Dyx1c1 and Dcdc2). In these experiments, neurons normally destined for the upper neocortical laminæ were transfected on embryonic day (E) 15.5, and we designed experiments to test whether these migration phenotypes were the result of targeting a specific type of projection neuron. We transfected litters with Dcdc2 shRNA, Dyx1c1 shRNA, Kiaa0319 shRNA, or fluorescent protein (as a control) at each of three gestational ages (E14.5, E15.5, or E16.5). Pups were allowed to come to term, and their brains were examined at 3 weeks of age for the position of transfected cells. We found that age of transfection did not affect the percentage of unmigrated neurons--transfection with Kiaa0319 shRNA resulted in heterotopia formation at all three ages. Overmigration of neurons transfected with Dcdc2 shRNA, while present following transfections at the later ages, did not occur following E14.5 transfections. These results are considered in light of the known functions of each of these candidate dyslexia susceptibility genes.