Biology subjects

Rajagopal, J.

Publications and source records attributed to Rajagopal, J..

11 recordsLinked to original sources

bioRxiv2026

Induced microfold cells are required for the function of pulmonary tertiary lymphoid organs that promote mucosal immunity

Tertiary lymphoid organs (TLOs) arise in response to infection and inflammation. We show that influenza-induced lung TLOs are comprised of inducible M cell (iM cell)-inducible bronchus-associated lymphoid tissue (iBALT) functional composites. iM cells are necessary for the appropriate architecture of adjacent immune follicles, which contain clonally expanded and sequence-diversified B cells. iM cell ablation results in fewer antigen-specific germinal center B cells, T follicular helper cells, and T helper memory cells, and a decrease in secretory IgA and IgG production accompanied by a loss of viral control. We find that the iM cells arise from epithelial progenitors that respond to a RANKL signal from infiltrating T cells, consistent with a model in which the patchy inflammation typical of pneumonia results in the unpredictable and varying distribution of coupled iM cells and adjacent iBALT. We propose that iM cells could be harnessed to promote infection control and enhance mucosal vaccination.

immunology↗

bioRxiv2025

YAP1 drives aggressive and therapy resistant state in melanoma through reprogramming the chromatin and regulating immune evasive programs

Despite promising initial results in targeting the RAF-MEK-ERK cascade, resistance to BRAF/MEK inhibitors remains a critical challenge in nearly 50% of melanoma patients. Our study demonstrates that robust YAP1 activation in metastatic melanoma correlates with poor survival and drives transcriptional programs linked to therapeutic resistance. Mechanistically, YAP1 predominantly remodels the chromatin landscape in resistant tumors by partnering with BRD4 and TEAD, creating a permissive transcriptional state that sustains oncogenic signaling. Clinical validation in biopsies from resistant melanoma confirms elevated expression of YAP1 target genes. Furthermore, pharmacological inhibition of BRD4 or TEAD reduces YAP1-driven transcription and reactivates antitumor immunity programs. TEAD specific inhibitors (and not verteporfin which is a highly non-specific inhibitor) synergize with immune checkpoint blockade in in vivo model system by promoting increased CD8 T cell infiltration and prolonged survival in the melanoma mouse model. Collectively, these findings reveal a chromatin-centric vulnerability in BRAF/MEK inhibitor-resistant melanoma and propose TEAD specific inhibitors as a promising dual strategy to overcome resistance and reinvigorate the immune response, offering a novel therapeutic avenue for patients. Resistance to BRAF and MEK inhibitors remains a major obstacle in the treatment of melanoma. Here, we show that elevated YAP1 activity in metastatic melanoma is associated with poor patient survival and drives transcriptional programs that promote therapeutic resistance. Mechanistically, YAP1 cooperates with BRD4 and TEAD to reprogram the chromatin landscape, establishing an oncogenic transcriptional state that sustains resistance. Analysis of patient-derived melanoma biopsies confirms increased expression of YAP1 target genes in resistant tumors. Targeting this epigenetic circuitry using pharmacological inhibitors of BRD4 or TEAD effectively suppresses YAP1-driven transcriptional output and restores antitumor immune activity, as evidenced by enhanced CD8 T cell infiltration and prolonged survival in resistant melanoma models. These effects are further supported by transcriptomic and chromatin accessibility analyses, which reveal reduced expression of immune-suppressive and proliferation-associated gene networks upon TEAD inhibition. Collectively, our findings identify a chromatin-centric mechanism underlying resistance to MAPK-targeted therapy and nominate TEAD inhibition as a promising dual-action strategy to both overcome resistance and re-engage antitumor immunity. This work offers a compelling therapeutic avenue for patients with drug-resistant melanoma.

cancer biology↗

bioRxiv2025

The identification and characterization of hillocks in the postnatal mouse and human airway

Adult lung regeneration has been deeply scrutinized over the past decade. In contrast, the injury resistance mechanisms of the neonatal and pediatric airway have received considerably less attention despite the manifest clinical importance. We recently reported the discovery of the airway hillock in adult murine and human airways. Adult hillocks are stratified structures with luminal squamous barrier cells overlying a dedicated basal stem cell population. Functionally, hillocks serve as an injury-resistant reservoir of dedicated hillock stem cells that can resurface and repopulate the airway epithelium after severe damage. Indeed, hillock basal stem cells undergo massive clonal expansion in the process of repopulating denuded airway epithelium. Since the postnatal lung encounters injuries that can result in airway epithelial denudation in the setting of respiratory infection or aspiration, we sought to assess whether hillocks are present in the neonatal airway. In this manuscript we identify and characterize hillocks in postnatal mouse and human pediatric airways. We show that hillocks are present in mice from postnatal day 3 onwards and that they expand in size through adulthood. The earliest hillocks are functionally immature, but they acquire their injury resistance properties over the course of postnatal maturation. By re-analyzing published pediatric scRNAseq data, we identify cells with a hillock squamous cell gene signature that is conserved across species. Finally, we identify bona fide hillocks in an 8-month-old infant and an 8-year-old child. We now wonder whether the presence and maturation of hillocks has implications for disorders of the neonatal and childhood airway. More specifically, we hypothesize that the incomplete maturation of hillocks could create a window of vulnerability in neonates who may be particularly susceptible to airway damage in the setting of infection or aspiration. Thus, there is a need to define when human hillocks first form and establish the time window during which they functionally mature as a prelude to determining whether the appearance and properties of hillocks correlate to clinical phenotypes and outcomes.

developmental biology↗

bioRxiv2024

Single-cell analysis of human airway epithelium identifies cell type-specific responses to Aspergillus and Coccidioides

Respiratory fungal infections pose a significant threat to human health. Animal models do not fully recapitulate human disease, necessitating advanced models to study human-fungal pathogen interactions. In this study, we utilized primary human airway epithelial cells (hAECs) to recapitulate the lung environment in vitro and investigate cellular responses to two diverse, clinically significant fungal pathogens, Aspergillus fumigatus and Coccidioides posadasii. To understand the mechanisms of early pathogenesis for both fungi, we performed single-cell RNA sequencing of infected hAECs. Analysis revealed that both fungi induced cellular stress and cytokine production. However, the cell subtypes affected and specific pathways differed between fungi, with A. fumigatus and C. posadasii triggering protein-folding-related stress in ciliated cells and hypoxia responses in secretory cells, respectively. This study represents one of the first reports of single-cell transcriptional analysis of hAECs infected with either A. fumigatus or C. posadasii, providing a vital dataset to dissect the mechanism of disease and potentially identify targetable pathways. ImportanceFungal infections in the lungs are dreaded complications for those with compromised immune systems and have limited treatment strategies available. These options are restricted further by the increased prevalence of treatment-resistant fungi. Many studies focus on how our immune systems respond to these pathogens, yet airway epithelial cells remain an understudied component of fungal infections in the lungs. Here, the authors provide a transcriptional analysis of primary human airway epithelial cells stimulated by two distinct fungal pathogens, Aspergillus fumigatus and Coccidioides posadasii. These data will enable further mechanistic studies of the contribution of the airway epithelium to initial host responses and represent a powerful new resource for investigators.

immunology↗

bioRxiv2024

Airway basal stem cells are necessary for the maintenance of functional intraepithelial airway macrophages.

Adult stem cells play a crucial role in tissue homeostasis and repair through multiple mechanisms. In addition to being able to replace aged or damaged cells, stem cells provide signals that contribute to the maintenance and function of neighboring cells. In the lung, airway basal stem cells also produce cytokines and chemokines in response to inhaled irritants, allergens, and pathogens, which affect specific immune cell populations and shape the nature of the immune response. However, direct cell-to-cell signaling through contact between airway basal stem cells and immune cells has not been demonstrated. Recently, a unique population of intraepithelial airway macrophages (IAMs) has been identified in the murine trachea. Here, we demonstrate that IAMs require Notch signaling from airway basal stem cells for maintenance of their differentiated state and function. Furthermore, we demonstrate that Notch signaling between airway basal stem cells and IAMs is required for antigen-induced allergic inflammation only in the trachea where the basal stem cells are located whereas allergic responses in distal lung tissues are preserved consistent with a local circuit linking stem cells to proximate immune cells. Finally, we demonstrate that IAM-like cells are present in human conducting airways and that these cells display Notch activation, mirroring their murine counterparts. Since diverse lung stem cells have recently been identified and localized to specific anatomic niches along the proximodistal axis of the respiratory tree, we hypothesize that the direct functional coupling of local stem cell-mediated regeneration and immune responses permits a compartmentalized inflammatory response.

cell biology↗

bioRxiv2023

A deep lung cell atlas reveals cytokine-mediated lineage switching of a rare cell progenitor of the human airway epithelium

The human airway contains specialized rare epithelial cells whose roles in respiratory disease are not well understood. Ionocytes express the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), while chemosensory tuft cells express asthma-associated alarmins. However, surprisingly, exceedingly few mature tuft cells have been identified in human lung cell atlases despite the ready identification of rare ionocytes and neuroendocrine cells. To identify human rare cell progenitors and define their lineage relationship to mature tuft cells, we generated a deep lung cell atlas containing 311,748 single cell RNA-Seq (scRNA-seq) profiles from discrete anatomic sites along the large and small airways and lung lobes of explanted donor lungs that could not be used for organ transplantation. Of 154,222 airway epithelial cells, we identified 687 ionocytes (0.45%) that are present in similar proportions in both large and small airways, suggesting that they may contribute to both large and small airways pathologies in CF. In stark contrast, we recovered only 3 mature tuft cells (0.002%). Instead, we identified rare bipotent progenitor cells that can give rise to both ionocytes and tuft cells, which we termed tuft-ionocyte progenitor cells (TIP cells). Remarkably, the cycling fraction of these TIP cells was comparable to that of basal stem cells. We used scRNA-seq and scATAC-seq to predict transcription factors that mark this novel rare cell progenitor population and define intermediate states during TIP cell lineage transitions en route to the differentiation of mature ionocytes and tuft cells. The default lineage of TIP cell descendants is skewed towards ionocytes, explaining the paucity of mature tuft cells in the human airway. However, Type 2 and Type 17 cytokines, associated with asthma and CF, diverted the lineage of TIP cell descendants in vitro, resulting in the differentiation of mature tuft cells at the expense of ionocytes. Consistent with this model of mature tuft cell differentiation, we identify mature tuft cells in a patient who died from an asthma flare. Overall, our findings suggest that the immune signaling pathways active in asthma and CF may skew the composition of disease-relevant rare cells and illustrate how deep atlases are required for identifying physiologically-relevant scarce cell populations.

cell biology↗

bioRxiv2023

Single Cell Transcriptomes, Lineage, and Differentiation of Functional Airway Microfold Cells

The airway epithelium is frequently exposed to pathogens and allergens, but the cells that are responsible for sampling these inhaled environmental agents have not been fully defined. Thus, there is a critical void in our understanding of how luminal antigens are delivered to the immune cells that drive the appropriate immune defenses against environmental assaults. In this study, we report the first single cell transcriptomes of airway Microfold (M) cells, whose gut counterparts have long been known for their antigen sampling abilities. Given their very recent discovery in the lower respiratory airways, the mechanisms governing the differentiation and functions of airway M cells are largely unknown. Here, we shed light on the pathways of airway M cell differentiation, establish their lineage, and identify a functional M cell-specific endocytic receptor, the complement receptor 2 (CR2). Lastly, we demonstrate that airway M cells can endocytose Aspergillus fumigatus conidia in a CR2-dependent manner. Collectively, this work lays a foundation for deepening our understanding of lung mucosal immunology and the mechanisms that drive lung immunity and tolerance.

cell biology↗

bioRxiv2023

Cross-kingdom anti-inflammatory effects of fungal melanin on airway epithelium by post-translational blockade of chemokine secretion

Respiratory infections caused by the human fungal pathogen Aspergillus fumigatus are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been fully defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of the chemokines CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction in neutrophil recruitment to the apical airway both in vitro and in vivo. Aspergillus-derived melanin, a major constituent of the fungal cell wall, dampened airway epithelial chemokine secretion in response to fungi, bacteria, and exogenous cytokines. Furthermore, melanin muted pathogen-mediated calcium fluxing and hindered actin filamentation. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens.

immunology↗

bioRxiv2022

Label free autofluorescence imaging permits comprehensive and simultaneous assignment of cell type identity and reveals the existence of airway secretory cell associated antigen passages (SAPs).

The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a properly orchestrated physiologic response. Thus, modern approaches to dissect physiologic mechanism would benefit from an ability to identify specific cell types in live tissues and image them in real time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the simultaneous study of 2 or 3 cell types. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in real time. Furthermore, we find that this methodology for direct cell type specific identification avoid potential pitfalls with the use of ostensibly cell type-specific markers that can be altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology using a clinically relevant model of cholinergic stimulation and identify dynamic secretory cell associated antigen passages (SAPs) that are highly reminiscent of previously reported goblet cell associated antigen passages (GAPs) in the intestine. eLifes Review ProcesseLife works to improve the process of peer review so that it more effectively conveys the assessment of expert reviewers to authors, readers and other interested parties. In the future we envision a system in which research is first published as a preprint and the outputs of peer review are the primary way research is assessed, rather than journal title. Our editorial process produces two outputs: i) an assessment by peers designed to be posted alongside a preprint for the benefit of the readers; i) detailed feedback on the manuscript for the authors, including requests for revisions and suggestions for improvement. Therefore we want to change how we construct and write peer reviews to make themuseful to both authors and readers in a way that better reflects the work you put into reading and thinking about a paper. eLife reviews now have three parts: O_LIAn evaluation summary (in two or three sentences) that captures the major conclusions of the review in a concise manner, accessible to a wide audience. C_LIO_LIA public review that details the strengths and weaknesses of the manuscript before you, and discusses whether the authors claims and conclusions are justified by their data. C_LIO_LIA set of private recommendations for the authors that outline how you think the science and its presentation could be strengthened. C_LI All three sections will be used as the basis for an eLife publishing decision, which will, as always, be made after a consultation among the reviewers and editor. Each of the public reviews will be published (anonymously) alongside the preprint, together with a response from the authors if they choose. In the case of papers we reject after review, the authors can choose to delay posting until their paper has been published elsewhere. If this is your first time going through this new process, we ask that you take some time to read our Reviewer Guide, which discusses how we see each section will be used, what it should contain, and what we hope it accomplishes. And we remind you that, with the shift of reviews from private correspondence to public discourse, it is more important than ever that reviews are written in a clear and constructive manner appropriate for a public audience and mindful of the impact language choices might have on the authors.

cell biology↗

bioRxiv2022

A quiescent resident progenitor pool is the central organizer of tendon healing

A tendons ordered extracellular matrix (ECM) is integral for transmitting force and highly prone to injury. How tendon cells, or tenocytes, embedded within this dense ECM mobilize and contribute to healing is unknown. Here, we identify a specialized Axin2+ population in mouse and human tendons that remains latent in homeostasis yet initiates the healing response and serves as a major source of tendon progenitors. Axin2+ tenocytes readily expand in vitro and express stem cell markers. In vivo, Axin2+-descendants are major functional contributors to repair: Axin2+ tenocytes proliferate, acquire injury responsive states, and re-adopt a tenocyte fate post-injury. Specific loss of Wnt secretion in Axin2+ cells alters their progenitor identity, disrupts their activation upon injury, and severely compromises any healing response. Our work highlights an unusual paradigm, wherein quiescent Axin2+ tenocytes self-regulate their identity and mobilization upon injury and provide the key initiating signal to organize a tendon-wide healing response.

cell biology↗

bioRxiv2020

CoolMPS™: Advanced massively parallel sequencing using antibodies specific to each natural nucleobase

Massively parallel sequencing (MPS) on DNA nanoarrays provides billions of reads at relatively low cost and enables a multitude of genomic applications. Further improvement in read length, sequence quality and cost reduction will enable more affordable and accurate comprehensive health monitoring tests. Currently the most efficient MPS uses dye-labeled reversibly terminated nucleotides (RTs) that are expensive to make and challenging to incorporate. Furthermore, a part of the dye-linker (scar) remains on the nucleobase after cleavage and interferes with subsequent sequencing cycles. We describe here the development of a novel MPS chemistry (CoolMPS) utilizing unlabeled RTs and four natural nucleobase-specific fluorescently labeled antibodies with fast (30 sec) binding. We implemented CoolMPS on MGIs PCR-free DNBSEQ MPS platform using arrays of 200nm DNA nanoballs (DNBs) generated by rolling circle replication and demonstrate 3-fold improvement in signal intensity and elimination of scar interference. Single-end 100-400 base and pair-end 2x150 base reads with high quality were readily generated with low out-of-phase incorporation. Furthermore, DNBs with less than 50 template copies were successfully sequenced by strong-signal CoolMPS with 3-times higher accuracy than in standard MPS. CoolMPS chemistry based on natural nucleobases has potential to provide longer, more accurate and less expensive MPS reads, including highly accurate "4-color sequencing" on the most efficient dye-crosstalk-free 2-color imagers with an estimated sequencing error rate of 0.00058% (one error in 170,000 base calls) in a proof-of-concept demonstration.

genomics↗