Gasdermin D promotes influenza virus-induced mortality through neutrophil amplification of inflammation.

Influenza virus activates cellular inflammasome pathways, which can be both beneficial and detrimental to infection outcomes. Here, we investigate the function of the inflammasome-activated, pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice (Gsdmd-/-) significantly attenuates influenza virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected Gsdmd-/- mice exhibit decreased inflammatory gene signatures shown by lung transcriptomics. Among these, diminished neutrophil gene activation signatures are corroborated by decreased detection of neutrophil elastase and myeloperoxidase in KO mouse lungs. Indeed, directly infected neutrophils are observed in vivo and infection of neutrophils in vitro induces release of DNA and tissue-damaging enzymes that is largely dependent on GSDMD. Neutrophil depletion in infected WT mice recapitulates the reductions in mortality, lung inflammation, and lung dysfunction observed in Gsdmd-/- animals, while depletion does not have additive protective effects in Gsdmd-/- mice. These findings implicate a function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a therapeutic avenue for treating severe influenza.

Luteolin prevents TNF-α-induced NF-κB activation and ROS production in cultured human placental explants and endothelial cells.

INTRODUCTION: Preeclampsia (PE) is a serious hypertensive pregnancy disorder and a leading cause of maternal and perinatal morbidity and mortality. Despite the prevalence and complications, there are no approved therapeutics to relieve PE symptoms. Inflammation, oxidative stress, and angiogenic imbalance have been shown to contribute to the PE pathophysiology, though there is a lack of understanding in how best to target these pathways in PE. We recently demonstrated that the bioflavonoid luteolin is a potent inhibitor of the anti-angiogenic and pro-hypertensive soluble fms-like tyrosine kinase 1 (sFlt-1), and here we aimed to determine if luteolin was also capable of reducing inflammation and oxidative stress pathways. METHODS: Tumor necrosis factor (TNF)-α, which is upregulated in PE, was utilized to stimulate these pathways in human placental explants and endothelial cells. Endothelin-1 (ET-1) and interleukin (IL)-6 in the media from explants and cells were measured via ELISA, and NF-κB localization and reactive oxygen species were detected via fluorescence microscopy. RESULTS: Pretreatment with luteolin demonstrated significant reductions in NF-κB activation, reactive oxygen species, superoxide, and IL-6 and ET-1 expression in endothelial cells. We also saw a significant reduction in phosphorylation of NF-κB in human placental explants. DISCUSSION: These data demonstrate that luteolin inhibits pathways implicated in the development of PE and should be explored further for its potential as a PE therapeutic.

Innate immune control of influenza virus interspecies adaptation.

Influenza virus pandemics are caused by viruses from animal reservoirs that adapt to efficiently infect and replicate in human hosts. Here, we investigated whether Interferon-Induced Transmembrane Protein 3 (IFITM3), a host antiviral factor with known human deficiencies, plays a role in interspecies virus infection and adaptation. We found that IFITM3-deficient mice and human cells could be infected with low doses of avian influenza viruses that failed to infect WT counterparts, identifying a new role for IFITM3 in controlling the minimum infectious viral dose threshold. Remarkably, influenza viruses passaged through Ifitm3-/- mice exhibited enhanced host adaptation, a result that was distinct from passaging in mice deficient for interferon signaling, which caused virus attenuation. Our data demonstrate that IFITM3 deficiency uniquely facilitates zoonotic influenza virus infections and subsequent adaptation, implicating IFITM3 deficiencies in the human population as a vulnerability for emergence of new pandemic viruses.

Bioflavonoid luteolin prevents sFlt-1 release via HIF-1α inhibition in cultured human placenta.

Preeclampsia (PE) is a serious hypertensive complication of pregnancy and is a leading cause of maternal death and major contributor to maternal and perinatal morbidity, including establishment of long-term complications. The continued prevalence of PE stresses the need for identification of novel treatments which can target prohypertensive factors implicated in the disease pathophysiology, such as soluble fms-like tyrosine kinase 1 (sFlt-1). We set out to identify novel compounds to reduce placental sFlt-1 and determine whether this occurs via hypoxia-inducible factor (HIF)-1α inhibition. We utilized a commercially available library of natural compounds to assess their ability to reduce sFlt-1 release from primary human placental cytotrophoblast cells (CTBs). Human placental explants from normotensive (NT) and preeclamptic (PE) pregnancies were treated with varying concentrations of luteolin. Protein and mRNA expression of sFlt-1 and upstream mediators were evaluated using ELISA, western blot, and real-time PCR. Of the natural compounds examined, luteolin showed the most potent inhibition of sFlt-1 release, with >95% reduction compared to vehicle-treated. Luteolin significantly inhibited sFlt-1 in cultured placental explants compared to vehicle-treated in a dose- and time-dependent manner. Additionally, significant decreases in HIF-1α expression were observed in luteolin-treated explants, suggesting a mechanism for sFlt-1 downregulation. The ability of luteolin to inhibit HIF-1α may be mediated through the Akt pathway, as inhibitors to Akt and its upstream regulator phosphatidylinositol-3 kinase (PI3K) resulted in significant HIF-1α reduction. Luteolin reduces anti-angiogenic sFlt-1 through inhibition of HIF-1α, making it a novel candidate for the treatment of PE.

Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice.

Interferon-induced transmembrane protein 3 (IFITM3) is an antiviral protein that alters cell membranes to block fusion of viruses. Conflicting reports identified opposing effects of IFITM3 on SARS-CoV-2 infection of cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with SARS-CoV-2 experience extreme weight loss and lethality compared to mild infection in wild-type (WT) mice. KO mice have higher lung viral titers and increases in inflammatory cytokine levels, immune cell infiltration, and histopathology. Mechanistically, we observe disseminated viral antigen staining throughout the lung and pulmonary vasculature in KO mice, as well as increased heart infection, indicating that IFITM3 constrains dissemination of SARS-CoV-2. Global transcriptomic analysis of infected lungs shows upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections in vivo.

Gasdermin D promotes influenza virus-induced mortality through neutrophil amplification of inflammation.

Influenza virus activates cellular inflammasome pathways, which can be either beneficial or detrimental to infection outcomes. Here, we investigated the role of the inflammasome-activated pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice significantly attenuated virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected GSDMD KO mice exhibited decreased inflammatory gene signatures revealed by lung transcriptomics, which also implicated a diminished neutrophil response. Importantly, neutrophil depletion in infected WT mice recapitulated the reduced mortality and lung inflammation observed in GSDMD KO animals, while having no additional protective effects in GSDMD KOs. These findings reveal a new function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a new therapeutic avenue for treating severe influenza.

Fertility Protection, A Novel Concept: Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Protect against Chemotherapy-Induced Testicular Cytotoxicity.

Currently, there is no viable option for fertility preservation in prepubertal boys. Experimentally, controlled vitrification of testicular tissue has been evaluated and found to cause potential structural damage to the spermatogonial stem cell (SSC) niche during cryopreservation. In this report, we leveraged the regenerative effect of human umbilical cord-derived Mesenchymal stem cell exosomes (h-UCMSC-Exo) to protect against testicular damage from the cytotoxic effects of polychemotherapy (CTX). A chemotherapy-induced testicular dysfunctional model was established by CTX treatment with cyclophosphamide and Busulfan in vitro (human Sertoli cells) and in prepubescent mice. We assessed the effects of the exosomes by analyzing cell proliferation assays, molecular analysis, immunohistochemistry, body weight change, serum hormone levels, and fertility rate. Our data indicates the protective effect of h-UCMSC-Exo by preserving the SSC niche and preventing testicular damage in mice. Interestingly, mice that received multiple injections of h-UCMSC-Exo showed significantly higher fertility rates and serum testosterone levels (p < 0.01). Our study demonstrates that h-UCMSC-Exo can potentially be a novel fertility protection approach in prepubertal boys triaged for chemotherapy treatment.

Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with an increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increased susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. We identified 1 rapalog (ridaforolimus) that was less potent in this regard and demonstrated that rapalogs promote spike-mediated entry into cells, by triggering the degradation of the antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increased virus entry inhibited mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitated its nuclear translocation and triggered microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity.

Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and immunothrombosis.

Severe acute respiratory syndrome coronavirus 2 (SARS­CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS­CoV-2 infections and that CASP4 expression correlates with severity of SARS­CoV-2 infection in humans. SARS­CoV-2­infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS­CoV-2­infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1ß, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS­CoV-2­induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

Growth differentiation factor-15 and its role in diabetes and cardiovascular disease.

Growth differentiation factor-15 (GDF-15) is cytokine involved in the regulation of multiple systems. Because it has regularly been shown to be increased in cardiovascular disease (CVD) and diabetes, it has been suggested that GDF-15 could be used as a biomarker for these diseases and their severity. However, several studies have demonstrated that GDF-15 has a protective role in regulation of inflammation, endothelial cell function, insulin sensitivity, weight gain, and is cardioprotective in myocardial infarction (MI). While GDF-15 has been implicated in the pathophysiology of many conditions including cancer, this review focuses on the potential functions of GDF-15 and signaling pathways implicated in its role regulating metabolism, insulin sensitivity, and the cardiovascular system.

Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice.

Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and antiviral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost minimal weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo , K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection of the lung and cardiovascular system, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice.

Differential regulation of sFlt-1 splicing by U2AF65 and JMJD6 in placental-derived and endothelial cells.

Despite years of study, the gestational disorder preeclampsia (PE) remains poorly understood. One proposed mechanism of PE development is increased soluble VEGF receptor-1 (sFlt-1), ultimately causing angiogenic imbalance and endothelial dysfunction. The soluble protein is an alternative splice variant of FLT1, which also encodes for the full-length receptor Flt-1. The mechanism of the alternative splicing, and the reason for its inappropriate increase in preeclampsia, is not well understood. U2 auxiliary factor 65 (U2AF65) and jumonji C domain-containing protein 6 (JMJD6) have been implicated in the splicing of sFlt-1. Using siRNA knockdown and plasmid overexpression in immortalized placental trophoblasts (BeWo) and primary endothelial cells (HUVECs), we examined the role these proteins play in production of sFlt-1. Our results showed that U2AF65 has little, if any, effect on sFlt-1 splicing, and JMJD6 may enhance sFlt-1 splicing, but is not necessary for splicing to occur. Utilizing a hypoxic environment to mimic conditions of the preeclamptic placenta, as well as examining placentae in the reduced uterine perfusion pressure (RUPP) model of PE, which exhibits increased circulating sFlt-1, we found increased expression of JMJD6 in both hypoxic cells and placental tissue. Additionally, we observed a potential role for U2AF65 and JMJD6 to regulate the extracellular matrix enzyme heparanase, which may be involved in the release of sFlt-1 protein from the extracellular matrix. It will be important to study the role of these proteins in different tissues in the future, as changes in expression had differential effects on sFlt-1 splicing in the different cell types studied here.

Biopolymer-Delivered, Maternally Sequestered NF-κB (Nuclear Factor-κB) Inhibitory Peptide for Treatment of Preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy that causes significant acute and long-term risk to the mother and the baby. The multifaceted maternal syndrome is driven by overproduction of circulating anti-angiogenic factors, widespread inflammation, and endothelial dysfunction. Nuclear factor-κB (NF-κB) is a transcription factor that plays a central role in the inflammatory response. Its activity is increased in the preeclamptic placenta, and it promotes the systemic endothelial dysfunction present in preeclampsia. There is an acute need for new therapeutics targeted to the causative pathways of preeclampsia. Our group has developed a drug delivery system based on the bioengineered protein ELP (elastin-like polypeptide) that is capable of stabilizing therapeutics in the maternal circulation and preventing their placental transfer. Here we used the ELP carrier system to deliver a peptide known to inhibit the NF-κB pathway. This polypeptide, containing a cell-penetrating peptide and an NF-κB inhibitory peptide derived from the p50 nuclear localization sequence (abbreviated SynB1-ELP-p50i), blocked NF-κB activation and prevented TNF-α (tumor necrosis factor alpha)-induced endothelin production in vitro. Fusion of the p50i peptide to the SynB1-ELP carrier slowed its plasma clearance and prevented its placental transfer in pregnant rats, resulting in increased deposition in the maternal kidney, liver, and placenta relative to the free peptide. When administered in a rat model of placental ischemia, SynB1-ELP-p50i partially ameliorated placental ischemia-induced hypertension and reduced placental TNF-α levels with no signs of toxicity. These data support the continued development of ELP-delivered NF-κB inhibitors as maternally sequestered anti-inflammatory agents for preeclampsia therapy.

Heparanase regulation of sFLT-1 release in trophoblasts in vitro.

INTRODUCTION: Preeclampsia is a common pregnancy disorder which is characterized by new onset hypertension and endothelial dysfunction. Despite efforts to determine the causal factors of this disease, little progress has been made in discerning the etiology. The hypoxic and ischemic placenta, however, is generally accepted as the source for secreted factors in the maternal circulation, such as sFLT-1, which drive the maternal syndrome. METHODS: Using BeWo placental trophoblast cells, we measured the role of hypoxia on sFLT-1 mRNA as well as protein production. We also exposed the cells to treatment with heparin and heparanase inhibitor OGT-2115. RESULTS: We found that under hypoxic conditions mRNA levels of sFLT-1 were unchanged compared to normoxic controls. Although the message level did not differ under hypoxic conditions, the sFLT-1 release into the media was significantly greater in hypoxia. Additionally, we found that sFLT-1 is able to bind heparan strands in the extracellular matrix with its heparin binding site. These heparan strands can be cleaved by the extracellular enzyme heparanase. We found that heparanase expression was significantly increased in hypoxia, and inhibiting the actions of heparanase attenuated the release of sFLT-1 into the media. DISCUSSION: While the placenta remains a source of sFLT-1, the mechanism of increased circulating sFLT-1 may differ than simple upregulation of the protein. These data demonstrate the potential importance of the role heparanase may play in releasing previously made sFLT-1 into the maternal circulation.

Acute Hypoxia and Chronic Ischemia Induce Differential Total Changes in Placental Epigenetic Modifications.

Preeclampsia is a common obstetrical complication, hallmarked by new-onset hypertension. Believed to result from placental insufficiency and chronic placental ischemia, the symptoms of preeclampsia are caused by release of pathogenic factors from the placenta itself, although the mechanisms of their regulation are in many cases unknown. One potential mechanism is through changes in placental epigenetic chromatin modifications, particularly histone acetylation and DNA methylation. Here, we determined the effects of chronic ischemia on global epigenetic modifications in the rodent placenta in vivo and acute hypoxia in BeWo placental trophoblast cells in vitro. Placental insufficiency via uterine artery restriction increased maternal blood pressure and fetal demise while decreasing placental and fetal mass. Global placental histone H3 acetylation levels were significantly decreased at H3 K9, K14, K18, K27, and K56. Interestingly, when BeWo-immortalized placental trophoblast cells were cultured in oxygen concentrations mimicking healthy and ischemic placentas, there was a significant increase in acetylated at K9, K18, K27, and K56. This was associated with a small but significant decrease in placental acetyl-CoA, suggesting depletion in the source of acetyl group donors. Finally, while global methylation of cytosine from placental DNA was low in both groups of animals (<1%), there was ∼50% increase in 5-mC in response to chronic ischemia. This suggests acute hypoxia and chronic ischemia induce differential global changes in histone acetylation in the placenta and that chronically altered metabolic profiles could affect histone acetylation in the placenta, thereby regulating production of pathogenic factors from the placenta during preeclampsia.

Pro-angiogenic therapeutics for preeclampsia.

Preeclampsia is a pregnancy-induced hypertensive disorder resulting from abnormal placentation, which causes factors such as sFlt-1 to be released into the maternal circulation. Though anti-hypertensive drugs and magnesium sulfate can be given in an effort to moderate symptoms, the syndrome is not well controlled. A hallmark characteristic of preeclampsia, especially early-onset preeclampsia, is angiogenic imbalance resulting from an inappropriately upregulated sFlt-1 acting as a decoy receptor binding vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), reducing their bioavailability. Administration of sFlt-1 leads to a preeclamptic phenotype, and several models of preeclampsia also have elevated levels of plasma sFlt-1, demonstrating its role in driving the progression of this disease. Treatment with either VEGF or PlGF has been effective in attenuating hypertension and proteinuria in multiple models of preeclampsia. VEGF, however, may have overdose toxicity risks that have not been observed in PlGF treatment, suggesting that PlGF is a potentially safer therapeutic option. This review discusses angiogenic balance as it relates to preeclampsia and the studies which have been performed in order to alleviate the imbalance driving the maternal syndrome.