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Extensive efforts have been made to study the human immune response following SARS-CoV-2 infection. Recent work by our lab provided insights into the molecular-level composition of the circulating IgG antibody repertoire evoked by SARS-CoV-2 primary infection and revealed key properties of the spike-directed antibodies and the epitopes they target in convalescent plasma (Voss et al., Science 2021). We showed that the antibody response in mild SARS-CoV-2 infection is directed to epitopes outside the RBD and primarily to the S2 domain of the spike glycoprotein. The continual emergence of globally circulating SARS-CoV-2 variants underscores the need for an ongoing study of the long-term evolution of our adaptive immune response to this virus. Collective knowledge about the molecular composition of human serological IgG memory to SARS-CoV-2 is limited. Our research uses single-cell sequencing and plasma IgG proteomics to better understand the evolving plasma antibody response to infection, vaccination, and breakthrough infection. In volunteers with a primary infection prior to vaccination, we can identify the serological recall of somatically mutated, pre-existing IgG antibodies targeting the S2 domain of the SARS-CoV-2 spike that cross-react with conserved epitopes expressed by endemic β-coronaviruses, indicating the existence of serological immune imprinting by prior infections. In contrast, in volunteers who received a full vaccine regimen and then had a breakthrough infection, the serological recall is markedly different—as shall be discussed. Collectively, these data reveal that the molecular nature of serological IgG recall appears to depend on the sequence and mode of exposure to SARS-CoV-2 spike antigen. Supported by NIH NCI COVID-19 SeroNet grant U54-CA260543 (R.S.B.)

SummaryType II nuclear hormone receptors, such as FXR, LXR, and PPAR, which function in glucose and lipid metabolism and serve as drug targets for metabolic diseases, are permanently positioned in the nucleus regardless of the ligand status. Ligand activation of these receptors is thought to occur by co-repressor/co-activator exchange, followed by initiation of transcription. 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 receptor binding. We show that chromatin accessibility, LXRα occupancy, and LXRα-dependent gene expression upon ligand activation require Foxa2. Unexpectedly, Foxa2 occupancy is drastically increased when LXRα is bound by an agonist. Our results suggest that Foxa2 and LXRα bind DNA as an interdependent complex during ligand activation. Our model requiring pioneering activity for ligand activation challenges the existing co-factor exchange mechanism and expands current understanding of nuclear receptor biology, suggesting that chromatin accessibility needs to be considered in design of drugs targeting nuclear receptors.

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.

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.

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-β-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-β-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.

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., Model describing establishment of H3K9me3/H3K14ac bivalent mark. In the first step, close residues H3K9 and H3K14 are acetylated by the same histone acetyltransferase (HAT). Then, H3K9 is deacetylated by Hdac3, allowing for subsequent triple methylation of H3K9 by Setdb1, which is found in complex with Kap1, and establishment of the bivalent region.

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.

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.

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.

Glioblastoma multiforme (GBM) is an aggressive malignant brain tumor with poor prognosis. Like other difficult-to-treat cancers, GBM utilizes glucose (glycolysis) at a high rate (the Warburg effect), but can adapt to stresses or starvation by using alternatives, including fatty acids or amino acids, as mitochondrial substrates. The unique features of GBM-specific metabolism may serve as novel therapeutic targets. Temozolomide (TMZ), along with surgery and radiation, are current standards for treating GBM. TMZ induces DNA damage, resulting in cell cycle arrest, and apoptosis. However, GBM can overcome TMZ by activating DNA repair enzymes. Our hypothesis is that combining TMZ with metabolic disrupting agents will potentiate TMZ cytotoxicity. To test this hypothesis, C6 (rat) and 263 (human) GBM cell lines were treated TMZ, with or without inhibitors of fatty acid oxidation or autophagy. Treated cells were stained and analyzed by flow cytometry for mitochondrial membrane potential (ΔΨ); uncoupling protein 2 (UCP2); Fas (CD95) and FasL (CD178); LC3A/B, detecting autophagy; and for DNA damage quantification. Explanted human GBM was stained for UCP2. We found that C6, 263, and explanted GBM tissue express significant levels of UCP2. In C6, etomoxir increased ΔΨ and decreased UCP2 expression. As predicted, etomoxir enhanced TMZ-induced DNA damage and the combination treatment elicited a greater response. Each treatment altered Fas and FasL expression, but the combination of etomoxir plus TMZ were necessary to achieve significant differences. Interestingly, we observed one high and one relatively low population of cells with regard to LC3A/B. However, treatment with etomoxir caused the largest increase in accumulation of LC3A/B, an indicator of autophagy. Finally, we determined the optimum combinations of combined drugs to achieve the greatest level of toxicity. We demonstrate that combination treatment using TMZ with metabolic inhibitors potentiates the effectiveness of TMZ on GBM.

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. Electronic supplementary material The online version of this article (doi:10.1186/s40478-014-0143-5) contains supplementary material, which is available to authorized users.

Glioblastoma multiforme (GBM) is an aggressive malignant brain tumor that has a very poor prognosis. In fact, the average life expectancy at the time of diagnosis is approximately 1.2 years. The cells that make up GBM, like cells in other difficult-to-treat cancers, use available glucose at a very high rate (high rate glycolysis, also known as the Warburg effect). The cells also exhibit metabolic plasticity allowing the growing tumor to quickly adapt to available nutrient even under conditions of starvation. In fact, during stress or starvation, GBM utilize autophagy, a process whereby the tumor cell engulfs its own organelles to fuel its energetic needs. Taken together, these unique features of GBM metabolism suggest that GBM-specific metabolic pathways could serve as novel therapeutic targets for GBM. Thus, the advantages of metabolic targets as therapies are at least two-fold: (1) the cancer cell can no longer use an efficient strategy for energy production, and (2) metabolic disruption results in stresses that can alter the cell surface and potentially lead to recognition of the danger by immune cells from the tumor microenvironment. Temozolomide (TMZ), the current standard-of-care therapy for treating GBM, was designed to cause DNA damage, cell cycle checkpoint arrest, oxidative stress, and apoptosis. However, some GBM overcome TMZ treatment by activating DNA repair enzymes, such as the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) or poly-ADP-ribose polymerase (PARP). In this study, we address the possibility that combination treatment using TMZ to promote DNA damage combined with an inhibitor of GBM-specific energy strategies (that may be required for effective DNA damage repair) will potentiate the effectiveness of TMZ alone.