Biology subjects

Wynn, E. A.

Publications and source records attributed to Wynn, E. A..

6 recordsLinked to original sources

bioRxiv2025

Spatial Heterogeneity of Macrophages in the Human Lung

RationaleTranscriptionally-defined populations of interstitial macrophages (IMs) and airspace macrophages (AMs) have recently been identified in the human lung. However, the anatomic locations occupied by these populations (i.e. alveoli, pleura, airways, or arteries) have not been fully defined. ObjectivesTo determine the distribution of transcriptionally-defined human macrophages in the major anatomical lung structures and to identify alterations in their distribution and programming induced by cigarette smoking. MethodsSingle-cell RNA sequencing was performed on lung tissue from eight human donors without pulmonary disease (four smokers and four nonsmokers). Microdissection was used to isolate distinct pulmonary anatomical structures from each lung: alveoli, pleura, airways, and arteries. Transcriptional profiles of subpopulations of interstitial macrophages (IMs) and alveolar macrophages (AMs) were analyzed based on their anatomical structure of origin and smoking status. Measurements and Main ResultsFive major IM and five AM subpopulations in human lungs are identified. We demonstrate significant differences in the accumulation patterns of each macrophage subset within anatomical structures, though each subset was detected in each. Immunofluorescent microscopy confirmed anatomical structure-specific accumulation patterns of IMs. ConclusionsIn this study, we highlight key differences in the accumulation of lung macrophage subpopulations in anatomical structures but find programming within macrophage subpopulations is largely conserved, regardless of structure of origin or smoking status. We also detect populations of inflammatory AMs and IMs which accumulate within the airways, but not the alveolar parenchyma, of human cigarette smokers. We introduce a novel three-tiered hierarchy nomenclature to distinguish transcriptionally defined human lung IM subsets as 1{degrees}) Monocyte-like vs Antigen Presenting, 2{degrees}) Quiescent vs Inflammatory, and 3{degrees}) FOLR2high vs FOLR2low. This study is the first to report the fractional accumulation of human lung macrophage subsets by lung anatomical structure. SummaryLung anatomical structure-specific single cell RNA sequencing is introduced to identify and determine the local composition of human lung leukocytes, including 5 populations of human interstitial macrophages.

immunology↗

bioRxiv2025

Deconvoluting drug interactions based on M. tuberculosis physiologic processes:Transcriptional disaggregation of the BPaL regimen in vivo

Identification of optimal antibiotic combination treatments for tuberculosis (TB) in preclinical studies is impeded by the limited information conventional pharmacodynamic (PD) markers provide about drug interactions. Measurement of individual drug activity based on colony forming units (CFU) does not reliably predict the activity of drug combinations, potentially because one drug may affect the physiology of Mycobacterium tuberculosis (Mtb) in a way that either favors or disfavors the activity of a second drug. SEARCH-TB is a novel candidate PD approach which uses targeted in vivo transcriptional profiling to evaluate the effects of drugs on Mtb physiology. To test the capacity of SEARCH-TB to elucidate drug interactions, we deconstructed the BPaL (bedaquiline, pretomanid, linezolid) regimen in the BALB/c high-dose aerosol mouse infection model, measuring the effect of 2, 7, and 14-day treatment with drugs in monotherapy, pairwise combinations, and as a 3-drug combination. Monotherapy rapidly induced drug-specific Mtb transcriptional responses by day 2 with continued evolution over 14 days. Bedaquiline dominated pairwise combinations with both pretomanid and linezolid. The pretomanid-linezolid combination gave a blended response, inducing transcriptional profiles "intermediate" between either drug. In the 3-drug BPaL regimen, the addition of both pretomanid and linezolid to bedaquiline yielded a greater transcriptional response than expected based on pairwise results. This work demonstrates that physiologic perturbations induced by a single drug may be modified in complex ways when drugs are combined. This establishes proof of concept that SEARCH-TB is a highly granular readout of drug interactions in vivo, providing information distinct from CFU burden and suggesting a future in which regimen selection is informed by in vivo molecular measures of Mtb physiology.

genomics↗

bioRxiv2025

Physiologic recovery of Mycobacterium tuberculosis from drug injury: A molecular study of post antibiotic effect in mice

Post-antibiotic effect (PAE) describes the delayed recovery of bacteria following antibiotic exposure. PAE is thought to underlie tuberculosis (TB) treatment forgiveness, i.e. the capacity of regimens to tolerate non-adherence. The basis of PAE in Mycobacterium tuberculosis (Mtb) remains poorly understood, partly because PAE has conventionally been measured based on change in Mtb burden in vitro rather than change in Mtb physiology in vivo. We investigated Mtb physiologic recovery in the BALB/c mouse model following sub-curative 2- and 4-week durations of the standard isoniazid, rifampin, pyrazinamide, ethambutol (HRZE) treatment. Measurement of rRNA synthesis via the RS ratio(R) and the entire transcriptome via SEARCH-TB elucidated the dynamics of physiologic recovery. Mtb burden did not increase over 28 days of drug-free post-treatment recovery, indicating prolonged PAE in vivo. The RS ratio indicated that Mtb ribosomal RNA synthesis resumed within four days of treatment interruption. However, transcriptional changes indicative of metabolic reactivation were delayed for over two weeks. Processes critical for replication, including expression of genes involved in protein and cell wall synthesis, remained suppressed throughout 28 days post-treatment. Longer treatment induced more extensive physiologic perturbation and was associated with slower and less complete recovery. Expression of processes that are typically induced by environmental stress (e.g., DosR regulon, universal stress proteins, and heat shock proteins) exhibited the reverse, decreasing during drug treatment and rising during recovery. These findings provide a new basis for understanding PAE based on drug-induced injury and physiologic recovery. Following relatively short durations of HRZE, physiologic recovery of Mtb was a slow, sequential and incomplete process in vivo. Our observation that longer treatment resulted in even slower recovery suggests that Mtb may progressively lose capacity to recover. This work establishes a tractable experimental framework for quantifying the forgiveness of new TB treatment regimens in vivo.

microbiology↗

bioRxiv2025

Simulating Longitudinal Single-cell RNA Sequencing Data with RESCUE

As single-cell RNA-sequencing (scRNA-seq) becomes more widely used in transcriptomic research, complex experimental designs, such as longitudinal studies, become increasingly feasible. Longitudinal scRNA-seq enables the study of transcriptomic changes over time within specific cell types, yet guidance on analytical approaches and resources for study planning, such as power analysis, remains limited. Data simulation is a valuable tool for evaluating analysis method performance and informing study design decisions, including sample size selection. Currently, most scRNA-seq simulation methods simulate cells for a single sample, thus ignoring the between-sample and between-subject variability inherent to longitudinal scRNA-seq data. Here, we introduce RESCUE (REpeated measures Single Cell RNA-seqUEncing data simulation), a novel method that simulates longitudinal scRNA-seq data using a gamma-Poisson frame-work and incorporates additional variability between samples and subjects. We demonstrate our methods ability to reproduce important data properties and demonstrate its application in study planning. RES-CUE is implemented as an R package and is available at https://github.com/ewynn610/RESCUE.

genomics↗

bioRxiv2024

Emergence of antibiotic-specific Mycobacterium tuberculosis phenotypes during prolonged treatment of mice

A major challenge in tuberculosis (TB) therapeutics is that antibiotic exposure leads to changes in the physiologic state of M. tuberculosis (Mtb) which may enable the pathogen to withstand treatment. While antibiotic-treated Mtb have been evaluated in short-term in vitro experiments, it is unclear if and how long-term in vivo treatment with diverse antibiotics with varying treatment-shortening activity (sterilizing activity) affect Mtb physiologic states differently. Here, we used SEARCH-TB, a pathogen-targeted RNA-sequencing platform, to characterize the Mtb transcriptome in the BALB/c high-dose aerosol infection mouse model following 4-week treatment with three sterilizing and three non-sterilizing antibiotics. Certain transcriptional changes were concordant among most antibiotics, including decreased expression of genes associated with protein synthesis and metabolism, and the induction of certain genes associated with stress responses. However, the magnitude of this concordant response differed between antibiotics. Sterilizing antibiotics rifampin, pyrazinamide, and bedaquiline generated a more quiescent Mtb state than did non-sterilizing antibiotics isoniazid, ethambutol, and streptomycin, as indicated by decreased expression of genes associated with translation, transcription, secretion of immunogenic proteins, metabolism, and cell wall synthesis. Additionally, we identified distinguishing transcriptional effects specific to each antibiotic, indicating that different mechanisms of action induce distinct patterns of cellular injury. In addition to elucidating Mtb physiologic changes associated with antibiotic stress, this study demonstrates the value of SEARCH-TB as a highly granular pharmacodynamic assay that reveals antibiotic effects that are not apparent based on culture alone.

genomics↗

bioRxiv2023

Transcriptional adaptation of drug-tolerant Mycobacterium tuberculosis in mice

Transcriptome evaluation of Mycobacterium tuberculosis in the lungs of laboratory animals during long-term treatment has been limited by extremely low abundance of bacterial mRNA relative to eukaryotic RNA. Here we report a targeted amplification RNA sequencing method called SEARCH-TB. After confirming that SEARCH-TB recapitulates conventional RNA-seq in vitro, we applied SEARCH-TB to Mycobacterium tuberculosis-infected BALB/c mice treated for up to 28 days with the global standard isoniazid, rifampin, pyrazinamide, and ethambutol regimen. We compared results in mice with 8-day exposure to the same regimen in vitro. After treatment of mice for 28 days, SEARCH-TB suggested broad suppression of genes associated with bacterial growth, transcription, translation, synthesis of rRNA proteins and immunogenic secretory peptides. Adaptation of drug-stressed Mycobacterium tuberculosis appeared to include a metabolic transition from ATP-maximizing respiration towards lower-efficiency pathways, modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pumps expression. Despite markedly different expression at pre-treatment baseline, murine and in vitro samples had broadly similar transcriptional change during treatment. The differences observed likely indicate the importance of immunity and pharmacokinetics in the mouse. By elucidating the long-term effect of tuberculosis treatment on bacterial cellular processes in vivo, SEARCH-TB represents a highly granular pharmacodynamic monitoring tool with potential to enhance evaluation of new regimens and thereby accelerate progress towards a new generation of more effective tuberculosis treatment.

microbiology↗