TOP1 inhibition therapy protects against SARS-CoV-2-induced lethal inflammation.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.

Accumulation of immune-suppressive CD4 + T cells in aging - tempering inflammaging at the expense of immunity.

The 'immune risk profile' has been shown to predict mortality in the elderly, highlighting the need to better understand age-related immune dysfunction. While aging leads to many defects affecting all arms of the immune system, this review is focused on the accrual of immuno-suppressive CD4 + T cell populations, including FoxP3 + regulatory T cells, and subsets of IL-10-producing T follicular helper cells. New data suggest that such accumulations constitute feedback mechanisms to temper the ongoing progressive low-grade inflammation that develops with age, the so-called "inflammaging", and by doing so, how they have the potential to promote healthier aging. However, they also impair effector immune responses, notably to infections, or vaccines. These studies also reinforce the idea that the aged immune system should not be considered as a poorly functional version of the young one, but more as a dynamic system in which CD4 + T cells, and other immune/non-immune subsets, differentiate, interact with their milieu and function differently than in young hosts. A better understanding of these unique interactions is thus needed to improve effector immune responses in the elderly, while keeping inflammaging under control.

maxATAC: Genome-scale transcription-factor binding prediction from ATAC-seq with deep neural networks.

Transcription factors read the genome, fundamentally connecting DNA sequence to gene expression across diverse cell types. Determining how, where, and when TFs bind chromatin will advance our understanding of gene regulatory networks and cellular behavior. The 2017 ENCODE-DREAM in vivo Transcription-Factor Binding Site (TFBS) Prediction Challenge highlighted the value of chromatin accessibility data to TFBS prediction, establishing state-of-the-art methods for TFBS prediction from DNase-seq. However, the more recent Assay-for-Transposase-Accessible-Chromatin (ATAC)-seq has surpassed DNase-seq as the most widely-used chromatin accessibility profiling method. Furthermore, ATAC-seq is the only such technique available at single-cell resolution from standard commercial platforms. While ATAC-seq datasets grow exponentially, suboptimal motif scanning is unfortunately the most common method for TFBS prediction from ATAC-seq. To enable community access to state-of-the-art TFBS prediction from ATAC-seq, we (1) curated an extensive benchmark dataset (127 TFs) for ATAC-seq model training and (2) built "maxATAC", a suite of user-friendly, deep neural network models for genome-wide TFBS prediction from ATAC-seq in any cell type. With models available for 127 human TFs, maxATAC is the largest collection of high-performance TFBS prediction models for ATAC-seq. maxATAC performance extends to primary cells and single-cell ATAC-seq, enabling improved TFBS prediction in vivo. We demonstrate maxATAC's capabilities by identifying TFBS associated with allele-dependent chromatin accessibility at atopic dermatitis genetic risk loci.

Accessible chromatin maps of inflammatory bowel disease intestine nominate cell-type mediators of genetic disease risk.

Inflammatory Bowel Disease ( IBD ) is a chronic and often debilitating autoinflammatory condition, with an increasing incidence in children. Standard-of-care therapies lead to sustained transmural healing and clinical remission in fewer than one-third of patients. For children, TNFα inhibition remains the only FDA-approved biologic therapy, providing an even greater urgency to understanding mechanisms of response. Genome-wide association studies ( GWAS ) have identified 418 independent genetic risk loci contributing to IBD, yet the majority are noncoding and their mechanisms of action are difficult to decipher. If causal, they likely alter transcription factor ( TF ) binding and downstream gene expression in particular cell types and contexts. To bridge this knowledge gap, we built a novel resource: multiome-seq (tandem single-nuclei ( sn )RNA-seq and chromatin accessibility ( snATAC )-seq) of intestinal tissue from pediatric IBD patients, where anti-TNF response was defined by endoscopic healing. From the snATAC-seq data, we generated a first-time atlas of chromatin accessibility (putative regulatory elements) for diverse intestinal cell types in the context of IBD. For cell types/contexts mediating genetic risk, we reasoned that accessible chromatin will co-localize with genetic disease risk loci. We systematically tested for significant co-localization of our chromatin accessibility maps and risk variants for 758 GWAS traits. Globally, genetic risk variants for IBD, autoimmune and inflammatory diseases are enriched in accessible chromatin of immune populations, while other traits (e.g., colorectal cancer, metabolic) are enriched in epithelial and stromal populations. This resource opens new avenues to uncover the complex molecular and cellular mechanisms mediating genetic disease risk.

Cholestasis alters polarization and suppressor function of hepatic regulatory T cells.

Fibrosing cholangiopathies, including biliary atresia and primary sclerosing cholangitis, involve immune-mediated bile duct epithelial injury and hepatic bile acid (BA) retention (cholestasis). Regulatory T-cells (Tregs) can prevent auto-reactive lymphocyte activation, yet the effects of BA on this CD4 lymphocyte subset are unknown. Gene regulatory networks for hepatic CD4 lymphocytes in a murine cholestasis model revealed Tregs are polarized to Th17 during cholestasis. Following bile duct ligation, Stat3 deletion in CD4 lymphocytes preserved hepatic Treg responses. While pharmacological reduction of hepatic BA in MDR2-/- mice prompted Treg expansion and diminished liver injury, this improvement subsided with Treg depletion. A cluster of patients diagnosed with biliary atresia showed both increased hepatic Treg responses and improved 2-year native liver survival, supporting that Tregs might protect against neonatal bile duct obstruction. Together, these findings suggest liver BA determine Treg function and should be considered as a therapeutic target to restore protective hepatic immune responses.

Elevated CD153 Expression on Aged T Follicular Helper Cells is Vital for B cell Responses.

Our recent data showed that an aberrant IL-10-producing T follicular helper population (Tfh10) accumulates dramatically with age and is associated with age-related declines in vaccine responsiveness. Through single cell gene expression and chromatin accessibility analysis of IL-10+ and IL-10- memory CD4+ T cells from young and aged mice, we identified increased expression of CD153 on aged Tfh and Tfh10 cells. Mechanistically, we linked inflammaging (increased IL-6 levels) to elevated CD153 expression of Tfh cells through c-Maf. Surprisingly, blockade of CD153 in aged mice significantly reduced their vaccine-driven antibody response, which was associated with decreased expression of ICOS on antigen-specific Tfh cells. Combined, these data show that an IL-6/c-Maf/CD153 circuit is critical for maintaining ICOS expression. Thus, although overall Tfh-mediated B cell responses are reduced in the context of vaccines and aging, our data suggest that elevated expression of CD153 on Tfh cells potentiates the remaining Tfh function in aged mice.

Alveolar epithelial progenitor cells require Nkx2-1 to maintain progenitor-specific epigenomic state during lung homeostasis and regeneration.

Lung epithelial regeneration after acute injury requires coordination cellular coordination to pattern the morphologically complex alveolar gas exchange surface. During adult lung regeneration, Wnt-responsive alveolar epithelial progenitor (AEP) cells, a subset of alveolar type 2 (AT2) cells, proliferate and transition to alveolar type 1 (AT1) cells. Here, we report a refined primary murine alveolar organoid, which recapitulates critical aspects of in vivo regeneration. Paired scRNAseq and scATACseq followed by transcriptional regulatory network (TRN) analysis identified two AT1 transition states driven by distinct regulatory networks controlled in part by differential activity of Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids was sufficient to cause transition to a proliferative stressed Krt8+ state, and AEP-specific deletion of Nkx2-1 in adult mice led to rapid loss of progenitor state and uncontrolled growth of Krt8+ cells. Together, these data implicate dynamic epigenetic maintenance via Nkx2-1 as central to the control of facultative progenitor activity in AEPs.

An atlas of gene regulatory networks for memory CD4 + T cells in youth and old age.

Aging profoundly affects immune-system function, promoting susceptibility to pathogens, cancers and chronic inflammation. We previously identified a population of IL-10-producing, T follicular helper-like cells (" Tfh10 "), linked to suppressed vaccine responses in aged mice. Here, we integrate single-cell ( sc )RNA-seq, scATAC-seq and genome-scale modeling to characterize Tfh10 - and the full CD4 + memory T cell ( CD4 + TM ) compartment - in young and old mice. We identified 13 CD4 + TM populations, which we validated through cross-comparison to prior scRNA-seq studies. We built gene regulatory networks ( GRNs ) that predict transcription-factor control of gene expression in each T-cell population and how these circuits change with age. Through integration with pan-cell aging atlases, we identified intercellular-signaling networks driving age-dependent changes in CD4 + TM. Our atlas of finely resolved CD4 + TM subsets, GRNs and cell-cell communication networks is a comprehensive resource of predicted regulatory mechanisms operative in memory T cells, presenting new opportunities to improve immune responses in the elderly.

Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates.

Perinatal inflammatory stress is associated with early life morbidity and lifelong consequences for pulmonary health. Chorioamnionitis, an inflammatory condition affecting the placenta and fluid surrounding the developing fetus, affects 25 to 40% of preterm births. Severe chorioamnionitis with preterm birth is associated with significantly increased risk of pulmonary disease and secondary infections in childhood, suggesting that fetal inflammation may markedly alter the development of the lung. Here, we used intra-amniotic lipopolysaccharide (LPS) challenge to induce experimental chorioamnionitis in a prenatal rhesus macaque (Macaca mulatta) model that mirrors structural and temporal aspects of human lung development. Inflammatory injury directly disrupted the developing gas exchange surface of the primate lung, with extensive damage to alveolar structure, particularly the close association and coordinated differentiation of alveolar type 1 pneumocytes and specialized alveolar capillary endothelium. Single-cell RNA sequencing analysis defined a multicellular alveolar signaling niche driving alveologenesis that was extensively disrupted by perinatal inflammation, leading to a loss of gas exchange surface and alveolar simplification, with notable resemblance to chronic lung disease in newborns. Blockade of the inflammatory cytokines interleukin-1ß and tumor necrosis factor-α ameliorated LPS-induced inflammatory lung injury by blunting stromal responses to inflammation and modulating innate immune activation in myeloid cells, restoring structural integrity and key signaling networks in the developing alveolus. These data provide new insight into the pathophysiology of developmental lung injury and suggest that modulating inflammation is a promising therapeutic approach to prevent fetal consequences of chorioamnionitis.

Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli.

In this study, we developed a dynamic mathematical model of E. coli cell-free protein synthesis (CFPS). Model parameters were estimated from a dataset consisting of glucose, organic acids, energy species, amino acids, and protein product, chloramphenicol acetyltransferase (CAT) measurements. The model was successfully trained to simulate these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the performance, e.g., the yield and rates of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity was most sensitive to the oxidative phosphorylation and glycolysis/gluconeogenesis pathways. Amino acid biosynthesis was also important for productivity, while overflow metabolism and TCA cycle affected the overall system state. In addition, translation was more important to productivity than transcription. Finally, CAT production was robust to allosteric control, as were most of the predicted metabolite concentrations; the exceptions to this were the concentrations of succinate and malate, and to a lesser extent pyruvate and acetate, which varied from the measured values when allosteric control was removed. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-free E. coli system, and could provide a foundation for genome scale, dynamic modeling of cell-free E. coli protein synthesis.

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

Improving designer glycan production in Escherichia coli through model-guided metabolic engineering.

Asparagine-linked (N-linked) glycosylation is the most common protein modification in eukaryotes, affecting over two-thirds of the proteome. Glycosylation is also critical to the pharmacokinetic activity and immunogenicity of many therapeutic proteins currently produced in complex eukaryotic hosts. The discovery of a protein glycosylation pathway in the pathogen Campylobacter jejuni and its subsequent transfer into laboratory strains of Escherichia coli has spurred great interest in glycoprotein production in prokaryotes. However, prokaryotic glycoprotein production has several drawbacks, including insufficient availability of non-native glycan precursors. To address this limitation, we used a constraint-based model of E. coli metabolism in combination with heuristic optimization to design gene knockout strains that overproduced glycan precursors. First, we incorporated reactions associated with C. jejuni glycan assembly into a genome-scale model of E. coli metabolism. We then identified gene knockout strains that coupled optimal growth to glycan synthesis. Simulations suggested that these growth-coupled glycan overproducing strains had metabolic imbalances that rerouted flux toward glycan precursor synthesis. We then validated the model-identified knockout strains experimentally by measuring glycan expression using a flow cytometric-based assay involving fluorescent labeling of cell surface-displayed glycans. Overall, this study demonstrates the promising role that metabolic modeling can play in optimizing the performance of a next-generation microbial glycosylation platform.

Sequence Specific Modeling of E. coli Cell-Free Protein Synthesis.

Cell-free protein synthesis (CFPS) is a widely used research tool in systems and synthetic biology. However, if CFPS is to become a mainstream technology for applications such as point of care manufacturing, we must understand the performance limits and costs of these systems. Toward this question, we used sequence specific constraint based modeling to evaluate the performance of E. coli cell-free protein synthesis. A core E. coli metabolic network, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid biosynthesis, and degradation was augmented with sequence specific descriptions of transcription and translation and effective models of promoter function. Model parameters were largely taken from literature; thus the constraint based approach coupled the transcription and translation of the protein product, and the regulation of gene expression, with the availability of metabolic resources using only a limited number of adjustable model parameters. We tested this approach by simulating the expression of two model proteins: chloramphenicol acetyltransferase and dual emission green fluorescent protein, for which we have data sets; we then expanded the simulations to a range of additional proteins. Protein expression simulations were consistent with measurements for a variety of cases. The constraint based simulations confirmed that oxidative phosphorylation was active in the CAT cell-free extract, as without it there was no feasible solution within the experimental constraints of the system. We then compared the metabolism of theoretically optimal and experimentally constrained CFPS reactions, and developed parameter free correlations which could be used to estimate productivity as a function of carbon number and promoter type. Lastly, global sensitivity analysis identified the key metabolic processes that controlled CFPS productivity and energy efficiency. In summary, sequence specific constraint based modeling of CFPS offered a novel means to a priori estimate the performance of a cell-free system, using only a limited number of adjustable parameters. While we modeled the production of a single protein in this study, the approach could easily be extended to multiprotein synthetic circuits, RNA circuits, or the cell-free production of small molecule products.