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Biology subjects

Carroll, M. C.

Publications and source records attributed to Carroll, M. C..

11 recordsLinked to original sources
bioRxiv2026

Complete biosynthesis of penicillin G in Nicotiana benthamiana

Commercial penicillin production has relied on microbial fermentation for more than 80 years. Here, we engineered the plant, Nicotiana benthamiana, to produce penicillin G in its leaves by transient expression of up to seven fungal biosynthetic genes. Remarkably, all recombinant proteins localize to the analogous subcellular compartments without engineering signal peptide sequences or post-translational modification sites. Although non-ribosomal peptide synthetases occur widely in fungi and bacteria to produce a plethora of specialized metabolites, their evolutionary distribution does not extend to plants. Our results now open a new metabolic frontier for natural product synthesis, and offer possibilities to address global health concerns through an alternative biotechnology platform for fungal-derived pharmaceutical production.

synthetic biology↗
bioRxiv2025

In situ profiling of plasma cell clonality with image-based single-cell transcriptomics

Image-based single-cell transcriptomics can identify diverse cell types within intact tissues. However, in adaptive immunity, V(D)J recombination generates unique immune receptors within cells of the same type, leading to important functional variation that is not yet defined by these methods. Here we introduce B-cell-receptor multiplexed error robust fluorescence in situ hybridization (BCR-MERFISH), which distinguishes plasma cell clones based on V-gene usage in combination with transcriptome profiling. We demonstrate that BCR-MERFISH accurately identifies V-gene usage in cell culture and in mice with restricted or native plasma cell diversity. We then use BCR-MERFISH to reveal the microbiota-dependent changes in plasma cell abundance, clonal diversity, and public clonotype usage in the mouse gut and the non-uniform distribution of plasma cell clones along the mouse ileum. As tissue context is an essential modulator of plasma cell dynamics, we anticipate that BCR-MERFISH may offer new insights into a wide range of immunological questions.

immunology↗
bioRxiv2024

Proton pump inhibitors modulate esophageal epithelial barrier function and crosstalk with eosinophils

BackgroundEosinophilic esophagitis (EoE) is a chronic allergic disease characterized by esophageal dysfunction, type-2 inflammation, and esophageal eosinophilic infiltrate. While proton pump inhibitor (PPI) therapy is commonly used for EoE management, the underlying mechanism of action remains unclear. MethodsAir-liquid interface culture of esophageal epithelial cells was employed to investigate the impact of the PPI omeprazole on barrier integrity in IL-13-treated cultures. Epithelial chemokine secretion was assessed following stimulation with IL-13 and omeprazole, and the migration of eosinophils from healthy human donors was evaluated using 3 {micro}m pore-sized transwells. A co-culture system of epithelial cells and eosinophils was employed to study chemokine secretion and eosinophil adhesion and activation markers. ResultsOmeprazole treatment in the IL-13-treated air-liquid interface (ALI) model resulted in 186 differentially expressed genes and restored barrier integrity compared to ALI treated with IL-13 alone. Omeprazole treatment reduced STAT6 phosphorylation, downregulated calpain 14, and upregulated desmoglein-1 in the IL-13-treated air-liquid interface samples. IL-13-induced upregulation of Eotaxin-3, CXCL10, and periostin, but this was downregulated by omeprazole. Further, the expression of CD11b, CD18, and CD69 was lower on eosinophils from omeprazole-treated epithelial-eosinophil co-cultures, which also had lower levels of eotaxin-3, CXCL10, CCL2, and CCL4. ConclusionOmeprazole reduced the effects of IL-13 in both the epithelial air-liquid interface model and eosinophil-epithelial co-cultures, reducing barrier dysfunction, chemokine expression, and upregulation of eosinophil adhesion markers.

immunology↗
bioRxiv2024

Interferon-γ signaling in eosinophilic esophagitis has implications for epithelial barrier function and programmed cell death.

ObjectiveEosinophilic esophagitis (EoE) is a chronic esophageal inflammatory disorder characterized by eosinophil-rich mucosal inflammation and tissue remodeling. Transcriptional profiling of esophageal biopsies has previously revealed upregulation of type I and II interferon (IFN) response genes. We aim to unravel interactions between immune and epithelial cells and examine functional significance in esophageal epithelial cells. Design We investigated epithelial gene expression from EoE patients using single-cell RNA sequencing and a confirmatory bulk RNA-sequencing experiment of isolated epithelial cells. The functional impact of interferon signaling on epithelial cells was investigated using in vitro organoid models. ResultsWe observe upregulation of interferon response signature genes (ISGs) in the esophageal epithelium during active EoE compared to other cell types, single-cell data, and pathway analyses, identified upregulation in ISGs in epithelial cells isolated from EoE patients. Using an esophageal organoid and air-liquid interface models, we demonstrate that IFN-{gamma} stimulation triggered disruption of esophageal epithelial differentiation, barrier integrity, and induced apoptosis via caspase upregulation. We show that an increase in cleaved caspase-3 is seen in EoE tissue and identify interferon gamma (IFNG) expression predominantly in a cluster of majority-CD8+ T cells with high expression of CD69 and FOS. ConclusionThese findings offer insight into the interplay between immune and epithelial cells in EoE. Our data illustrate the relevance of several IFN-{gamma}-mediated mechanisms on epithelial function in the esophagus, which have the potential to impact epithelial function during inflammatory conditions. Key MessagesWhat is already known about this topic: O_LIThe transcriptome of esophageal biopsy tissue reproducibly distinguishes eosinophilic esophagitis from histologically normal tissue, with evidence of mixed inflammatory signals. C_LIO_LIInterferon response signature genes are elevated in EoE biopsy tissue compared to controls, suggesting T1 in addition to T2 cytokine signaling within EoE mucosa. C_LI What this study adds: O_LIWe observe reproducible, robust upregulation of interferon signature genes in esophageal epithelium, and we confirm that esophageal epithelium expresses functional IFN-a and IFN-{gamma} receptors. C_LIO_LIIFN-{gamma} treatment of epithelial organoids has several detrimental effects, including decreased proliferation, organoid formation, and increased caspase activation. C_LIO_LIAnalysis of single-cell RNA-sequencing data from of EoE biopsy tissue during active disease and remission identified that a CD8+ population expressing high levels of FOS, ITGAE, and ITGA1 expresses high levels of IFNG C_LI How this study might affect research, practice, or policy: O_LIWe identify esophageal epithelium as the cellular source for interferon response gene signature in EoE and a CD8+ tissue-resident memory T-cell population was the main source of IFNG. C_LIO_LIFurther mechanistic studies are required to identify how non-T2 signaling mechanisms like IFN-{gamma} signaling contribute to EoE pathogenesis, and if this pathway can be targeted as an adjunctive therapy for EoE. C_LI

immunology↗
bioRxiv2024

Lupus-associated innate receptors drive extrafollicular evolution of autoreactive B cells

In systemic lupus erythematosus, recent findings highlight the extrafollicular (EF) pathway as prominent origin of autoantibody-secreting cells (ASCs). CD21loCD11c+ B cells, associated with aging, infection, and autoimmunity, are contributors to autoreactive EF ASCs but have an obscure developmental trajectory. To study EF kinetics of autoreactive B cell in tissue, we adoptively transferred WT and gene knockout B cell populations into the 564Igi mice - an autoreactive host enriched with autoantigens and T cell help. Time-stamped analyses revealed TLR7 dependence in early escape of peripheral B cell tolerance and establishment of a pre-ASC division program. We propose CD21lo cells as precursors to EF ASCs due to their elevated TLR7 sensitivity and proliferative nature. Blocking receptor function reversed CD21 loss and reduced effector cell generation, portraying CD21 as a differentiation initiator and a possible target for autoreactive B cell suppression. Repertoire analysis further delineated proto-autoreactive B cell selection and receptor evolution toward self-reactivity. This work elucidates receptor and clonal dynamics in EF development of autoreactive B cells, and establishes modular, native systems to probe mechanisms of autoreactivity.

immunology↗
bioRxiv2023

Spatial and temporal profiling of the complement system uncovered novel functions of the alternative complement pathway in brain development

Mounting evidence implicated the classical complement pathway (CP) in normal brain development, and the pathogenesis of neuropsychiatric and neurodegenerative diseases. However the source and regulation of complement in the brain remain unclear. Using MERFISH, a spatial transcriptomic method with single-cell resolution, we established a developmental brain atlas of the complement system. We showed that the brain synthesizes essential building blocks of the complement system locally with remarkable cellular and spatial heterogeneity. We provided transcriptional evidence supporting the presence of the alternative pathway (AP), but lack of lectin pathway (LP) activity in postnatal brain development. Cell type, temporal and spatial expression patterns of genes involved indicate non-redundant functions of the CP and AP. In addition, deficiency in Masp3-driven AP resulted in developmental and cognitive defects, indicating essential functions of the AP, an observation that highlights the necessity to disentangle differential involvement of the three complement activation pathways in development and diseases.

neuroscience↗
bioRxiv2023

The impact of prenatal alcohol and synthetic cannabinoid exposure on behavioral adaptations in adolescent offspring and alcohol self-administration in adulthood

Prenatal exposure to alcohol or cannabinoids can produce enduring neurobiological, cognitive, and behavioral changes in the offspring. Furthermore, prenatal co-exposure to alcohol and cannabinoids induces malformations in brain regions associated with reward and stress-related circuitry. This study examined the effects of co-exposure to alcohol and the synthetic cannabinoid (SCB) CP55,940 throughout gastrulation and neurulation in rats on basal corticosterone levels and a battery of behavioral tests during adolescence and alcohol self-administration in adulthood. Importantly, we find that prenatal alcohol exposure (PAE) caused lower baseline corticosterone levels in adolescent males and females. Co-exposure to alcohol + CP produced hyperactivity during open field test in males, but not females. During the two-bottle choice alcohol-drinking procedure, prenatal cannabinoid exposed male and female adolescent rats drank more alcohol than their vehicle-exposed controls. In adulthood, female rats treated with prenatal cannabinoid exposure (PCE), showed an overall total increase in alcohol intake during alcohol self-administration; but this was not found in males. When the reinforcer was changed to a 1% sucrose solution, male rats exposed to PCE, showed a reduced self-administration compared to vehicle-exposed males, potentially indicative of an anhedonic response. This lower self-administration persisted when 20% alcohol was reintroduced to the sucrose solution. Lastly, following an abstinence period, there were no changes due to prenatal drug exposure in either males or females. Overall, these data suggest lasting consequences of prenatal alcohol and cannabinoid exposure during adolescence and adulthood in male and female rats.

animal behavior and cognition↗
bioRxiv2023

Invasion of spontaneous germinal centers by naive B cells is rapid and persistent

In autoreactive germinal centers (GC) initiated by a single rogue B cell clone, wild-type B cells expand and give rise to clones that target other autoantigens, known as epitope spreading. The chronic, progressive nature of epitope spreading calls for early interventions, but the kinetics and molecular requirements for wild-type B cell invasion and participation in GC remain largely unknown. With parabiosis and adoptive transfer approaches in a murine model of systemic lupus erythematosus, we demonstrate that wild-type B cells join existing GCs rapidly, clonally expand, persist, and contribute to autoantibody production and diversification. The invasion of autoreactive GCs required TLR7, B cell receptor specificity, antigen presentation, and type I interferon signaling. The adoptive transfer model provides a novel tool for identifying early events in the breaking of B cell tolerance in autoimmunity. One Sentence SummaryThe autoreactive germinal center is an open structure that is susceptible to rapid and persistent naive B cell invasion, with clonal expansion and auto-antibody induction and diversification.

immunology↗
bioRxiv2022

Complex subsets but redundant clonality after B cells egress from spontaneous germinal centers

Affinity matured self-reactive antibodies are found in autoimmune diseases like systemic lupus erythematous. Here we used fate-mapping reporter mice and single cell transcriptomics coupled to antibody repertoire analysis to characterize the post-germinal center (GC) B cell compartment in a new mouse model of autoimmunity. Antibody secreting cells (ASCs) and memory B cells (MemBs) from spontaneous GCs grouped into multiple subclusters. ASCs matured into two terminal clusters, with distinct secretion, antibody repertoire and metabolic profiles. MemBs contained FCRL5+ and CD23+ subsets, with different in vivo localization in the spleen. GC-derived FCRL5+ MemBs share transcriptomic and repertoire properties with atypical B cells found in aging and infection and localize to the marginal zone, suggesting a similar contribution to recall responses. While transcriptomically diverse, ASC and MemB subsets maintained an underlying clonal redundancy. Therefore, self-reactive clones could escape subset-targeting therapy by perpetuation of self-reactivity in distinct subsets.

immunology↗
bioRxiv2019

Toll-like receptor 2 stimulation augments esophageal epithelial barrier integrity

BackgroundA key concept of the hygiene hypothesis is that the microbiome modulates both epithelial barrier integrity as well as host immune responses. Defective expression of tight junction complex proteins alters this homeostatic process, and plays a role in atopic disorders including eosinophilic esophagitis. We tested the hypothesis that Toll-like receptor 2 (TLR2) stimulation improves esophageal barrier function in a cell-intrinsic manner by upregulation of TJ-protein expression using an in vitro model of human epithelium. MethodsPattern recognition receptor expression was assessed in esophageal epithelial cells from patients with EoE and non-EoE control patients. Functional consequences of TLR2 stimulation were investigated using human esophageal EPC2-hTERT cells in the three-dimensional air-liquid interface culture (ALI) model to evaluate transepithelial electrical resistance (TEER) and FITC-Dextran permeability. Characterization of TLR2-stimulated ALI cultures was performed by histology, immunohistochemistry, western blotting and chromatin immunoprecipitation. ResultsTLR2 stimulation increased TEER (1.28 to 1.31-fold) and decreased paracellular permeability to FITC-Dextran. Notably, TLR2 stimulation-induced increases in TEER were abolished by treatment with anti-TLR2 blocking antibody. Tight junction complex proteins claudin 1 and zonula occludens 1 were increased following TLR2 stimulation, and chromatin immunoprecipitation analysis demonstrated significant increase in histone 4 acetyl binding at the CLDN1 enhancer and promoter following zymosan treatment, implying the occurrence of durable chromatin changes in the esophageal epithelium. ConclusionsOur findings reveal that the TLR2 pathway may play a regulatory role as a mechanism that maintains epithelial barrier homeostasis in the esophagus.

immunology↗