Hybrid immunity to SARS-CoV-2 arises from serological recall of IgG antibodies distinctly imprinted by infection or vaccination.

We used plasma IgG proteomics to study the molecular composition and temporal durability of polyclonal IgG antibodies triggered by ancestral SARS-CoV-2 infection, vaccination, or their combination ("hybrid immunity"). Infection, whether primary or post-vaccination, mainly triggered an anti-spike antibody response to the S2 domain, while vaccination predominantly induced anti-RBD antibodies. Immunological imprinting persisted after a secondary (hybrid) exposure, with >60% of the ensuing serological response originating from the initial antibodies generated during the first exposure. We highlight one instance where hybrid immunity arising from breakthrough infection resulted in a marked increase in the breadth and affinity of a highly abundant vaccination-elicited plasma IgG antibody, SC27. With an intrinsic binding affinity surpassing a theoretical maximum (K D < 5 pM), SC27 demonstrated potent neutralization of various SARS-CoV-2 variants and SARS-like zoonotic viruses (IC 50 ∼0.1-1.75 nM) and provided robust protection in vivo . Cryo-EM structural analysis unveiled that SC27 binds to the RBD class 1/4 epitope, with both VH and VL significantly contributing to the binding interface. These findings suggest that exceptionally broad and potent antibodies can be prevalent in plasma and can largely dictate the nature of serological neutralization. HIGHLIGHTS: ▪ Infection and vaccination elicit unique IgG antibody profiles at the molecular level▪ Immunological imprinting varies between infection (S2/NTD) and vaccination (RBD)▪ Hybrid immunity maintains the imprint of first infection or first vaccination▪ Hybrid immune IgG plasma mAbs have superior neutralization potency and breadth.

Integrative analyses highlight functional regulatory variants associated with neuropsychiatric diseases.

Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types. Expression-gene mapping, network analyses and chromatin looping nominated candidate disease-relevant target genes modulated by these daSNVs. Follow-up integration of daSNV gene editing with clinical cohort analyses suggested that magnesium transport dysfunction may increase neuropsychiatric disease risk and indicated that common genetic pathomechanisms may mediate specific symptoms that are shared across multiple neuropsychiatric diseases.

Mendelian randomization and pathway analysis demonstrate shared genetic associations between lupus and coronary artery disease.

Coronary artery disease (CAD) is a leading cause of death in patients with systemic lupus erythematosus (SLE). Despite clinical evidence supporting an association between SLE and CAD, pleiotropy-adjusted genetic association studies are limited and focus on only a few common risk loci. Here, we identify a net positive causal estimate of SLE-associated non-HLA SNPs on CAD by traditional Mendelian randomization (MR) approaches. Pathway analysis using SNP-to-gene mapping followed by unsupervised clustering based on protein-protein interactions (PPIs) identifies biological networks composed of positive and negative causal sets of genes. In addition, we confirm the casual effects of specific SNP-to-gene modules on CAD using only SNP mapping to each PPI-defined functional gene set as instrumental variables. This PPI-based MR approach elucidates various molecular pathways with causal implications between SLE and CAD and identifies biological pathways likely causative of both pathologies, revealing known and novel therapeutic interventions for managing CAD in SLE.

Pioneer factor Foxa2 enables ligand-dependent activation of type II nuclear receptors FXR and LXRα.

OBJECTIVE: Type II nuclear hormone receptors, including farnesoid X receptors (FXR), liver X receptors (LXR), and peroxisome proliferator-activated receptors (PPAR), which serve as drug targets for metabolic diseases, are permanently positioned in the nucleus and thought to be bound to DNA regardless of the ligand status. However, recent genome-wide location analysis showed that LXRα and PPARα binding in the liver is largely ligand-dependent. We hypothesized that pioneer factor Foxa2 evicts nucleosomes to enable ligand-dependent binding of type II nuclear receptors and performed genome-wide studies to test this hypothesis. METHODS: ATAC-Seq was used to profile chromatin accessibility; ChIP-Seq was performed to assess transcription factors (Foxa2, FXR, LXRα, and PPARα) binding; and RNA-Seq analysis determined differentially expressed genes in wildtype and Foxa2 mutants treated with a ligand (GW4064 for FXR, GW3965, and T09 for LXRα). RESULTS: We reveal that chromatin accessibility, FXR binding, LXRα occupancy, and ligand-responsive activation of gene expression by FXR and LXRα require Foxa2. Unexpectedly, Foxa2 occupancy is drastically increased when either receptor, FXR or LXRα, is bound by an agonist. In addition, co-immunoprecipitation experiments demonstrate that Foxa2 interacts with either receptor in a ligand-dependent manner, suggesting that Foxa2 and the receptor, bind DNA as an interdependent complex during ligand activation. Furthermore, PPARα binding is induced in Foxa2 mutants treated with FXR and LXR ligands, leading to the activation of PPARα targets. CONCLUSIONS: Our model requires pioneering activity for ligand activation that challenges the existing ligand-independent binding mechanism. We also demonstrate that Foxa2 is required to achieve activation of the proper receptor - one that binds the added ligand - by repressing the activity of a competing receptor.

Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver.

Post-translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We identified a bivalent combination, a dually marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent sequential ChIP-Seq identified dually marked single nucleosome regions, with reduced number of sites and decreased signal in old livers, confirming mass spectrometry results. We detected H3K9me3 and H3K14ac bulk ChIP-Seq signal in reChIP nucleosome regions, suggesting a correlation between H3K9me3/H3K14ac bulk bivalent genomic regions and dually marked single nucleosomes. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied both bulk and single nucleosome bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.

Hypothalamus-Pituitary-Adrenal Dysfunction in Cholestatic Liver Disease.

The Hypothalamic-Pituitary-Adrenal (HPA) axis has an important role in maintaining the physiological homeostasis in relation to external and internal stimuli. The HPA axis dysfunctions were extensively studied in neuroendocrine disorders such as depression and chronic fatigue syndrome but less so in hepatic cholestasis, cirrhosis or other liver diseases. The HPA axis controls many functions of the liver through neuroendocrine forward signaling pathways as well as negative feedback mechanisms, in health and disease. This review describes cell and molecular mechanisms of liver and HPA axis physiology and pathology. Evidence is presented from clinical and experimental model studies, demonstrating that dysfunctions of HPA axis are correlated with liver cholestatic disorders. The functional interactions of HPA axis with the liver and immune system in cases of bacterial and viral infections are also discussed. Proinflammatory cytokines stimulate glucocorticoid (GC) release by adrenals but they also inhibit bile acid (BA) efflux from liver. Chronic hepatic inflammation leads to cholestasis and impaired GC metabolism in the liver, so that HPA axis becomes depressed. Recently discovered interactions of GC with self-oscillating transcription factors that generate circadian rhythms of gene expression in brain and liver, in the context of GC replacement therapies, are also outlined.

Gulf war illness-related chemicals increase CD11b/c+ monocyte infiltration into the liver and aggravate hepatic cholestasis in a rodent model.

Gulf War Illness (GWI) is a chronic multisymptom disorder affecting veterans of the 1990-91 Gulf war. GWI was linked with exposure to chemicals including the nerve gas prophylactic drug pyridostigmine-bromide (PB) and pesticides (DEET, permethrin). Veterans with GWI exhibit prolonged, low-level systemic inflammation, though whether this impacts the liver is unknown. While no evidence exists that GWI-related chemicals are hepatotoxic, the prolonged inflammation may alter the liver's response to insults such as cholestatic injury. We assessed the effects of GWI-related chemicals on macrophage infiltration and its subsequent influence on hepatic cholestasis. Sprague Dawley rats were treated daily with PB, DEET and permethrin followed by 15 minutes of restraint stress for 28 days. Ten weeks afterward, GWI rats or naïve age-matched controls underwent bile duct ligation (BDL) or sham surgeries. Exposure to GWI-related chemicals alone increased IL-6, and CD11b+F4/80- macrophages in the liver, with no effect on biliary mass or hepatic fibrosis. However, pre-exposure to GWI-related chemicals enhanced biliary hyperplasia and fibrogenesis caused by BDL, compared to naïve rats undergoing the same surgery. These data suggest that GWI patients could be predisposed to developing worse liver pathology due to sustained low-level inflammation of the liver when compared to patients without GWI.

Direct Comparison of the Thioacetamide and Azoxymethane Models of Type A Hepatic Encephalopathy in Mice.

Acute liver failure is a devastating consequence of hepatotoxic liver injury that can lead to the development of hepatic encephalopathy. There is no consensus on the best model to represent these syndromes in mice, and therefore the aim of this study was to classify hepatic and neurological consequences of azoxymethane- and thioacetamide-induced liver injury. Azoxymethane-treated mice were euthanized at time points representing absence of minor and significant stages of neurological decline. Thioacetamide-treated mice had tissue collected at up to 3 days following daily injections. Liver histology, serum chemistry, bile acids, and cytokine levels were measured. Reflexes, grip strength measurement, and ataxia were calculated for all groups. Brain ammonia, bile acid levels, cerebral edema, and neuroinflammation were measured. Finally, in vitro and in vivo assessments of blood-brain barrier function were performed. Serum transaminases and liver histology demonstrate that both models generated hepatotoxic liver injury. Serum proinflammatory cytokine levels were significantly elevated in both models. Azoxymethane-treated mice had progressive neurological deficits, while thioacetamide-treated mice had inconsistent neurological deficits. Bile acids and cerebral edema were increased to a higher degree in azoxymethane-treated mice, while cerebral ammonia and neuroinflammation were greater in thioacetamide-treated mice. Blood-brain barrier permeability exists in both models but was likely not due to direct toxicity of azoxymethane or thioacetamide on brain endothelial cells. In conclusion, both models generate acute liver injury and hepatic encephalopathy, but the requirement of a single injection and the more consistent neurological decline make azoxymethane treatment a better model for acute liver failure with hepatic encephalopathy.

FXR-Mediated Cortical Cholesterol Accumulation Contributes to the Pathogenesis of Type A Hepatic Encephalopathy.

BACKGROUND & AIMS: Hepatic encephalopathy is a serious neurologic complication of acute and chronic liver diseases. We previously showed that aberrant bile acid signaling contributes to the development of hepatic encephalopathy via farnesoid X receptor (FXR)-mediated mechanisms in neurons. In the brain, a novel alternative bile acid synthesis pathway, catalyzed by cytochrome p450 46A1 (Cyp46A1), is the primary mechanism by which the brain regulates cholesterol homeostasis. The aim of this study was to determine if FXR activation in the brain altered cholesterol homeostasis during hepatic encephalopathy. METHODS: Cyp7A1-/- mice or C57Bl/6 mice pretreated with central infusion of FXR vivo morpholino, 2-hydroxypropyl-ß-cyclodextrin, or fed a cholestyramine-supplemented diet were injected with azoxymethane (AOM). Cognitive and neuromuscular impairment as well as liver damage and expression of Cyp46A1 were assessed using standard techniques. The subsequent cholesterol content in the frontal cortex was measured using commercially available kits and by Filipin III and Nile Red staining. RESULTS: There was an increase in membrane-bound and intracellular cholesterol in the cortex of mice treated with AOM that was associated with decreased Cyp46A1 expression. Strategies to inhibit FXR signaling prevented the down-regulation of Cyp46A1 and the accumulation of cholesterol. Treatment of mice with 2-hydroxypropyl-ß-cyclodextrin attenuated the AOM-induced cholesterol accumulation in the brain and the cognitive and neuromuscular deficits without altering the underlying liver pathology. CONCLUSIONS: During hepatic encephalopathy, FXR signaling increases brain cholesterol and contributes to neurologic decline. Targeting cholesterol accumulation in the brain may be a possible therapeutic target for the management of hepatic encephalopathy.

Glucocorticoids Cause Gender-Dependent Reversal of Hepatic Fibrosis in the MDR2-Knockout Mouse Model.

Hepatic cholestasis is associated with a significant suppression of the hypothalamus-pituitary-adrenal axis (HPA). In the present study, we tested the hypothesis that activation of the HPA axis by corticosterone treatment can reverse liver inflammation and fibrosis in a multidrug resistance protein 2 knockout (MDR2KO) transgenic mouse model of hepatic cholestasis. Friend Virus B NIH-Jackson (FVBN) control and MDR2KO male and female mice were treated with vehicle or corticosterone for two weeks, then serum and liver analyses of hepatic cholestasis markers were performed. Indicators of inflammation, such as increased numbers of macrophages, were determined. MDR2KO mice had lower corticotropin releasing hormone and corticosterone levels than FVBN controls in the serum. There was a large accumulation of CD68 and F4/80 macrophages in MDR2KO mice livers, which indicated greater inflammation compared to FVBNs, an effect reversed by corticosterone treatment. Intrahepatic biliary duct mass, collagen deposition and alpha smooth muscle actin (αSMA) were found to be much higher in livers of MDR2KO mice than in controls; corticosterone treatment significantly decreased these fibrosis markers. When looking at the gender-specific response to corticosterone treatment, male MDR2KO mice tended to have a more pronounced reversal of liver fibrosis than females treated with corticosterone.

Bile Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Promotes Neuroinflammation during Hepatic Encephalopathy in Mice.

Hepatic encephalopathy (HE) is a neuropsychiatric complication that occurs due to deteriorating hepatic function and this syndrome influences patient quality of life, clinical management strategies and survival. During acute liver failure, circulating bile acids increase due to a disruption of the enterohepatic circulation. We previously identified that bile acid-mediated signaling occurs in the brain during HE and contributes to cognitive impairment. However, the influences of bile acids and their downstream signaling pathways on HE-induced neuroinflammation have not been assessed. Conjugated bile acids, such as taurocholic acid (TCA), can activate sphingosine-1-phosphate receptor 2 (S1PR2), which has been shown to promote immune cell infiltration and inflammation in other models. The current study aimed to assess the role of bile-acid mediated S1PR2 signaling in neuroinflammation and disease progression during azoxymethane (AOM)-induced HE in mice. Our findings demonstrate a temporal increase of bile acids in the cortex during AOM-induced HE and identified that cortical bile acids were elevated as an early event in this model. In order to classify the specific bile acids that were elevated during HE, a metabolic screen was performed and this assay identified that TCA was increased in the serum and cortex during AOM-induced HE. To reduce bile acid concentrations in the brain, mice were fed a diet supplemented with cholestyramine, which alleviated neuroinflammation by reducing proinflammatory cytokine expression in the cortex compared to the control diet-fed AOM-treated mice. S1PR2 was expressed primarily in neurons and TCA treatment increased chemokine ligand 2 mRNA expression in these cells. The infusion of JTE-013, a S1PR2 antagonist, into the lateral ventricle prior to AOM injection protected against neurological decline and reduced neuroinflammation compared to DMSO-infused AOM-treated mice. Together, this identifies that reducing bile acid levels or S1PR2 signaling are potential therapeutic strategies for the management of HE.

Hepatic alterations are accompanied by changes to bile acid transporter-expressing neurons in the hypothalamus after traumatic brain injury.

Annually, there are over 2 million incidents of traumatic brain injury (TBI) and treatment options are non-existent. While many TBI studies have focused on the brain, peripheral contributions involving the digestive and immune systems are emerging as factors involved in the various symptomology associated with TBI. We hypothesized that TBI would alter hepatic function, including bile acid system machinery in the liver and brain. The results show activation of the hepatic acute phase response by 2 hours after TBI, hepatic inflammation by 6 hours after TBI and a decrease in hepatic transcription factors, Gli 1, Gli 2, Gli 3 at 2 and 24 hrs after TBI. Bile acid receptors and transporters were decreased as early as 2 hrs after TBI until at least 24 hrs after TBI. Quantification of bile acid transporter, ASBT-expressing neurons in the hypothalamus, revealed a significant decrease following TBI. These results are the first to show such changes following a TBI, and are compatible with previous studies of the bile acid system in stroke models. The data support the emerging idea of a systemic influence to neurological disorders and point to the need for future studies to better define specific mechanisms of action.

Traumatic brain injury causes selective, CD74-dependent peripheral lymphocyte activation that exacerbates neurodegeneration.

INTRODUCTION: Traumatic brain injury (TBI), a significant cause of death and disability, causes, as in any injury, an acute, innate immune response. A key component in the transition between innate and adaptive immunity is the processing and presentation of antigen by professional antigen presenting cells (APCs). Whether an adaptive immune response to brain injury is beneficial or detrimental is not known. Current efforts to understand the contribution of the immune system after TBI have focused on neuroinflammation and brain-infiltrating immune cells. Here, we characterize and target TBI-induced expansion of peripheral immune cells that may act as potential APCs. Because MHC Class II-associated invariant peptide (CLIP) is important for antigen processing and presentation, we engineered a competitive antagonist (CAP) for CLIP, and tested the hypothesis that peptide competition could reverse or prevent neurodegeneration after TBI. RESULTS: We show that after fluid percussion injury (FPI), peripheral splenic lymphocytes, including CD4+ and CD8+ T cells, regulatory T cells (Tregs), and γδ T cells, are increased in number within 24 hours after FPI. These increases were reversed by CAP treatment and this antagonism of CLIP also reduced neuroinflammation and neurodegeneration after TBI. Using a mouse deficient for the precursor of CLIP, CD74, we observed decreased peripheral lymphocyte activation, decreased neurodegeneration, and a significantly smaller lesion size following TBI. CONCLUSION: Taken together, the data support the hypothesis that neurodegeneration following TBI is dependent upon antigen processing and presentation that requires CD74.