Biology subjects

Brody, S. L.

Publications and source records attributed to Brody, S. L..

At least 19 recordsLinked to original sources

bioRxiv2025

Atg16l1 promotes lung transplant tolerance by regulating glycolysis in macrophages

Lung transplant survival is limited by the development of chronic lung allograft dysfunction (CLAD), a type of graft rejection that lacks effective treatments. Autophagy plays a crucial role in maintaining cellular homeostasis. In a single-nucleotide polymorphism screen, we found that lung recipients with two copies of a common hypofunctional genetic variant of autophagy-related 16-like 1 rs2241880 (ATG16L1T300A/T300A), known to deplete this protein from macrophages, were more likely to develop early CLAD. To understand this, we used a mouse orthotopic lung transplant model. Recipients encoding myeloid cell-specific deletion of Atg16l1 (Atg16l1{Delta}/{Delta}) or who harbor an engineered orthologous mutation (Atg16l1T316A/T316A) showed similar susceptibility to CLAD. Transcript profiling and mitochondrial tracking studies indicated that increased mitochondrial damage and decreased autophagic removal of mitochondria in Atg16l1-deficient macrophages were associated with heightened activation of the hypoxia-inducible factor 1 (Hif1) pathway and accumulation of glycolytic transcripts. Metabolic analysis revealed reduced oxidative phosphorylation, increased glycolytic activity, and higher IL-1{beta} expression in Atg16l1-deficient macrophages. Notably, the development of CLAD in Atg16l1{Delta}/{Delta} lung recipients could be significantly prevented by additionally deleting Hif1 in myeloid cells or by treating with the glycolysis inhibitor 2-deoxyglucose. Our results show how a common autophagy-related genetic variant disrupts macrophage metabolism and impairs lung transplant tolerance, pointing toward potential therapeutic strategies to combat CLAD.

immunology↗

bioRxiv2025

A stem cell-based platform for functional analysis of genetic variants in lung disease

Advances in genetic and transcriptomic technologies have identified large numbers of genes and variants of potential importance to human disease. Determining the function of these genes and variants is a critical bottleneck in understanding disease etiology. Variants of uncertain significance (VUS) are highly prevalent in our genomes, but our ability to identify them significantly outpaces our ability to determine their molecular and clinical consequences. We developed a genetically tractable induced pluripotent stem cell (iPSC) based platform to investigate gene variant pathogenicity in lung disease, using primary ciliary dyskinesia (PCD) as a model. We identified an individual with a clinical diagnosis of PCD and a VUS in the gene Multiciliate differentiation and DNA synthesis associated cell cycle protein (MCIDAS). Through gene-editing of iPSC-derived airway basal stem cells (iBCs), we precisely defined the molecular and cellular pathogenicity of the variant providing a successful application of the iPSC system to diagnose a lung disease.

cell biology↗

bioRxiv2025

Latent transforming growth factor binding protein-2 (LTBP2), an IPF biomarker of clinical decline, promotes TGF-beta signaling and lung fibrosis in mice

The identification of clinically predictive serum biomarkers for pulmonary fibrosis is a significant challenge and important goal. Multiple recent proteomic biomarker studies have identified latent transforming growth factor binding protein-2 (LTBP2) as a circulating factor associated with disease progression in fibrotic lung diseases in humans (including IPF), but its role in the development of fibrosis is incompletely defined. LTBP2 competes with the large latent transforming growth factor-beta (TGF{beta}) complex (LLC) for binding to the N-terminus of fibrillin and is thought to promote the release of active TGF{beta}. We hypothesized that LTBP2 deficiency would promote LLC sequestration in matrix and reduce TGF{beta} signaling. We recently reported an LTBP2 knockout (Ltbp2-/-) mouse with no baseline lung abnormalities. Here we show that Ltbp2-/- mice exposed to either bleomycin or silica have a significant reduction in fibrosis compared to wild type controls. Consistent with reduced fibrosis, after bleomycin Ltbp2-/- mouse lungs have reduced TGF{beta} signaling and isolated fibroblasts from Ltbp2-/-mice exhibit impaired migration in an in vitro wound closure assay. Transcriptomic analysis of bleomycin-treated control and Ltbp2-/- mouse lung tissue identified multiple LTBP2-regulated genes, including the lncRNA antisense of IGFR2 non-coding RNA (Airn) which has reported antifibrotic effects. Interestingly, we also observed that Ltbp2-/- mice had impaired epithelial repair after bleomycin treatment, a phenotype that also occurred in a naphthalene model of club cell injury. These findings provide evidence that LTBP2 is profibrotic and facilitates TGF{beta} signaling but is also required for normal airway epithelial repair.

pathology↗

bioRxiv2025

Ciliated cells promote high infectious potential of influenza A virus through the efficient intracellular activation of hemagglutinin

Influenza viruses utilize host proteases to activate the viral fusion protein, hemagglutinin (HA), into its fusion-competent form. Although proteolytic activation of HA is essential for virus replication, the cell-type dependence of HA activation within the airway epithelium and the subcellular location(s) in which it occurs are not well-established. To address these questions, we investigated the proteolytic activation of HA in differentiated human airway epithelial cells using contemporary and historical H1N1 and H3N2 strains. We find that activation is efficient across viral strains and subtypes but depends on cellular tropism, with ciliated cells activating HA more effectively than non-ciliated cells. Similar to prior observations in immortalized cell lines, we find that HA activation occurs intracellularly, constraining the antiviral activity of host-directed protease inhibitors. These results establish that HA activation within the airway epithelium depends on cellular tropism, and identify important considerations for the development of protease inhibitors as antivirals. ImportanceInfluenza entry requires the proteolytic activation of the viral fusion protein, hemagglutinin (HA). Activation occurs as new viruses are produced by infected cells. Efficient proteolytic activation is critical for viral pathogenesis, and inhibiting the requisite proteases could provide an effective host-directed antiviral strategy. To understand cellular constraints on HA activation and its sensitivity to inhibitors, we use complementary approaches to investigate these processes in differentiated airway epithelial cells. We find that ciliated cells activate HA with higher efficiency than non-ciliated cell types, establishing a new mechanism through which cellular tropism and virus infectious potential are connected. We also establish that HA activation begins in the Golgi, which may contribute to the limited the efficacy of inhibitors we observe despite their high in vitro potency.

microbiology↗

bioRxiv2025

AggreBots: configuring CiliaBots through guided, modular tissue aggregation

Ciliated biobots, or CiliaBots, are a class of engineered multicellular tissues that are capable of self-actuated motility propelled by the motile cilia located on their exterior surface. Correlations have been observed between CiliaBot motility patterns and their morphology and cilia distribution. However, precise control of these structural parameters to generate desired motility patterns predictably remains lacking. Here, we developed a novel Aggregated CiliaBot (AggreBot) platform capable of producing designer motility patterns through spatially controlled aggregation of epithelial spheroids made from human airway cells (referred to as CiliaBot Building Blocks or CBBs), yielding AggreBots with configurable geometry and distribution of active cilia. Guided multi-CBB aggregation led to the production of rod-, triangle-, and diamond-shaped AggreBots, which consistently effected greater motility than traditional single-spheroid CiliaBots. Furthermore, CBBs were found to maintain internal boundaries post-aggregation through the combined action of pathways controlling cellular fluidity and tissue polarity. This boundary fidelity, combined with the use of CBBs with immotile cilia due to mutations in the CCDC39 gene, allowed for the generation of hybrid AggreBots with precision control over the coverage and distribution of active cilia, further empowering control of motility patterns. Our results demonstrate the potential of AggreBots as self-propelling biological tissues through the establishment of morphological "levers" by which alterations to tissue motility can be theoretically planned and experimentally verified.

bioengineering↗

bioRxiv2024

Cilia dysfunction in the lateral ventricles after neonatal intraventricular hemorrhage does not lead to functional changes in cilia-based CSF flow networks

Intraventricular hemorrhage (IVH) has long been thought to lead to motile cilia dysfunction whereby intraventricular blood breakdown products damage and slough cilia from the ependymal wall. However, specifically how IVH may affect cilia development, structure, and transcriptional activation is not well-understood. Moreover, the impact of blood breakdown product-mediated cilia damage on the functional organization of cilia-based CSF flow networks is unknown. Here, we show hemoglobin exposure affects the number of ciliated ependymal cells in the lateral ventricle (LV) but does not impact in vitro beat frequency of the remaining cilia. Ultrastructurally, IVH decreases the total number of ciliary tufts without impacting axoneme structure. IVH does not result in changes in the expression of cilia-related genes and instead leads to downregulation of neurogenesis markers in parallel with innate immune upregulation. Functionally, we identify three previously uncharacterized cilia-mediated CSF flow domains in the LV lateral wall and show that IVH does not result in widespread disruption of their functional organization. These data de-emphasize cilia as a major contributor to global CSF dysfunction after IVH, and instead call attention to preserving the neurodevelopmental environment and preventing runaway innate immune system activation, as considerations to developing treatment strategies to prevent posthemorrhagic hydrocephalus and other neurodevelopmental sequelae.

neuroscience↗

bioRxiv2024

A first-in-kind MAPK13 inhibitor that can correct stem cell reprogramming and post-injury disease

The stress kinase MAPK13 (aka p38delta-MAPK) is an attractive entry point for therapeutic intervention because it regulates the structural remodeling that can develop after epithelial injury in the lung and likely other tissue sites. However, a selective, safe, and effective MAPK13 inhibitor is not yet available for experimental or clinical application. Here we identify a first-in-kind MAPK13 inhibitor using structure-based drug design combined with a screening funnel for cell safety and molecular specificity. In a mouse model of severe respiratory viral infection, treatment with this inhibitor (formulated as NuP-4A for intravenous use or Nu4-B for inhaled delivery) did not influence recovery from acute infectious illness, but still down-regulated basal-epithelial stem cell (basal-ESC) hyperplasia/metaplasia and in turn airway inflammation, mucus production, and pathophysiology biomarkers of chronic lung disease. Treatment prevented and reversed disease readouts equivalently to Mapk13 gene-knockout, and this benefit persisted after stopping treatment as a sign of disease modification. Further, NuP-4 treatment even at pM levels directly blocked basal-ESC reprogramming endpoints in organoid and cell-culture models derived from non-disease control and asthma subjects. The results thereby provide a new tool compound and drug candidate for basal-ESC reprogramming towards muco-obstructive lung diseases like asthma and any overlap with COPD and related diseases that depend on overactivity of MAPK13.

immunology↗

bioRxiv2024

MAPK13 controls structural remodeling and disease after epithelial injury

All living organisms are charged with repair after injury particularly at epithelial barrier sites, but in some cases this response leads instead to structural remodeling and long-term disease. Identifying the molecular and cellular control of this divergence is key to disease modification. In that regard, stress kinase control of epithelial stem cells is a rational entry point for study. Here we examine the potential for mitogen-activated protein kinase 13 (MAPK13) regulation of epithelial stem cells using models of respiratory viral injury and post-viral lung disease that resembles asthma. We show that Mapk13 gene-knockout mice handle acute infectious illness as expected but are protected against basal-epithelial stem cell (basal-ESC) hyperplasia and mucous metaplasia. In corresponding organoid models, Mapk13-deficiency directly controls the basal-ESC program for stemness endpoints of hyperplasia and mucous metaplasia. Extension to human studies shows induction/activation of MAPK13 in basal-epithelial cells in lung tissue samples from asthma and COPD patients. Further, in human organoid models, MAPK13 mRNA knockdown regulates basal-ESC stemness similarly to mouse models. Together, the data identify MAPK13 as a control point for structural remodeling after epithelial injury and a suitable target for down-regulation as a disease-modifying strategy.

immunology↗

bioRxiv2024

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.

cell biology↗

bioRxiv2024

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

cell biology↗

bioRxiv2024

mTOR signaling regulates aberrant epithelial cell proliferative and migratory behaviors characteristic of airway mucous metaplasia in asthma

In asthma, the airway epithelium is hyperplastic, hypertrophied, and lined with numerous large MUC5AC-containing goblet cells (GC). Furthermore, the normal epithelial architecture is disorganized with numerous, what we here describe as, ectopic goblet cells (eGC) deep within the thickened epithelial layer disconnected from the lumenal surface. mTOR is a highly conserved pathway that regulates cell size and proliferation. We hypothesized that the balance between mTOR and autophagy signaling regulates key features of the asthma epithelial layer. Airway histological sections from subjects with asthma had increased frequency of eGC and increased levels of mTOR phosphorylation target-Ribosomal S6. Using human airway epithelial cells (hAECs) with IL-13 stimulation and timed withdrawal to stimulate resolution, we found that multiple key downstream phosphorylation targets downstream from the mTOR complex were increased during early IL-13-mediated mucous metaplasia, and then significantly declined during resolution. The IL-13-mediated changes in mTOR signaling were paralleled by morphologic changes with airway epithelial hypertrophy, hyperplasia, and frequency of eGC. We then examined the relationship between mTOR and autophagy using mice deficient in autophagy protein Atg16L1. Despite having increased cytoplasmic mucins, mouse AECs from Atg16L1 deficient mice had no significant difference in mTOR downstream signaling. mTOR inhibition with rapamycin led to a loss of IL-13-mediated epithelial hypertrophy, hyperplasia, ectopic GC distribution, and reduction in cytoplasmic MUC5AC levels. mTOR inhibition was also associated with a reduction in aberrant IL-13-mediated hAEC proliferation and migration. Our findings demonstrate that mTOR signaling is associated with mucous metaplasia and is crucial to the disorganized airway epithelial structure and function characteristic of muco-obstructive airway diseases such as asthma. Graphical Abstract Key ConceptsO_LIThe airway epithelium in asthma is disorganized and characterized by cellular proliferation, aberrant migration, and goblet cell mucous metaplasia. C_LIO_LImTOR signaling is a dynamic process during IL-13-mediated mucous metaplasia, increasing with IL-13 stimulation and declining during resolution. C_LIO_LImTOR signaling is strongly increased in the asthmatic airway epithelium. C_LIO_LImTOR signaling is associated with the development of key features of the metaplastic airway epithelium including cell proliferation and ectopic distribution of goblet cells and aberrant cellular migration. C_LIO_LIInhibition of mTOR leads to decreased epithelial hypertrophy, reduced ectopic goblet cells, and cellular migration. C_LI

cell biology↗

bioRxiv2024

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.

microbiology↗

bioRxiv2023

A new MAPK13-guided inhibitor for respiratory inflammation and mucus production

Common respiratory diseases continue to represent a major public health problem, and much of the morbidity and mortality is due to airway inflammation and mucus production. Previous studies indicated a role for mitogen-activated protein kinase 14 (MAPK14) in this type of disease, but clinical trials are unsuccessful to date. Our previous work identified a related but distinct kinase known as MAPK13 that is activated in respiratory airway diseases and is required for mucus production in human cell-culture models. Support for MAPK13 function in these models came from effectiveness of MAPK13 versus MAPK14 gene-knockdown and from first-generation MAPK13-14 inhibitors. However, these first-generation inhibitors were incompletely optimized for blocking activity and were untested in vivo. Here we report the next generation and selection of a potent MAPK13-14 inhibitor (designated NuP-3) that more effectively down-regulates type-2 cytokine-stimulated mucus production in air-liquid interface and organoid cultures of human airway epithelial cells. We also show that NuP-3 treatment prevents respiratory airway inflammation and mucus production in new minipig models of airway disease triggered by type-2 cytokine challenge or respiratory viral infection. The results thereby provide the next advance in developing a small-molecule kinase inhibitor to address key features of respiratory disease. New and noteworthyThis study describes the discovery of a potent MAPK13-14 inhibitor and its effectiveness in models of respiratory airway disease. The findings thereby provide a scheme for pathogenesis and therapy of lung diseases (e.g., asthma, COPD, Covid-19, post-viral and allergic respiratory disease) and related conditions that implicate MAPK13-14 function. The findings also refine a hypothesis for epithelial and immune cell functions in respiratory disease that features MAPK13 as a possible component of this disease process.

immunology↗

bioRxiv2023

Foxj1 controls olfactory ciliogenesis and differentiation program of the olfactory sensory neurons

In vertebrates, olfactory receptors localize on multiple cilia elaborated on dendritic knobs of olfactory sensory neurons (OSNs). Although olfactory cilia dysfunction can cause anosmia, how their differentiation is programmed at the transcriptional level has remained largely unexplored. We discovered in zebrafish and mice that Foxj1, a forkhead domain-containing transcription factor linked with motile cilia biogenesis, is expressed in OSNs and required for olfactory epithelium (OE) formation. In keeping with the immotile nature of olfactory cilia, we observed that ciliary motility genes are repressed in zebrafish, mouse, and human OSNs. Strikingly, we also found that besides ciliogenesis, Foxj1 controls the differentiation of the OSNs themselves by regulating their cell type-specific gene expression, such as that of olfactory marker protein (omp) involved in odor-evoked signal transduction. In line with this, response to bile acid, an odor detected by OMP-positive OSNs, was significantly diminished in foxj1 mutant zebrafish. Taken together, our findings establish how the canonical Foxj1-mediated motile ciliogenic transcriptional program has been repurposed for the biogenesis of immotile olfactory cilia and for development of the OSNs.

neuroscience↗

bioRxiv2023

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies. Brief SummaryA mouse model of human DNAAF5 primary ciliary dyskinesia variants reveals gene dosage effects of mutant alleles and tissue-specific molecular requirements for cilia motor assembly.

cell biology↗

bioRxiv2022

Single cell RNA-seq identifies inflammation-induced loss of CFTR-expressing airway ionocytes in non-eosinophilic asthma

Asthma is the most common chronic airways disease worldwide and the severe treatment resistant subtype of asthma is responsible for the majority of disease burden. Asthma is heterogeneous in nature and can be classified according to airway infiltrates as eosinophilic or non-eosinophilic (sometimes referred to as Type 2 low), which is further divided into paucigranulocytic (low levels of granulocytes), or neutrophilic asthma characterized by elevated neutrophils, and mixed Type 1 and Type 17 cytokines in airway tissue, sputum, and bronchoalveolar lavage. Severe non-eosinophilic asthma currently has fewer effective treatment options and many of these patients fail to qualify for newer biologic monoclonal therapies. The cystic fibrosis transmembrane conductance regulator (CFTR) is a key protein whose function is dysregulated in multiple respiratory diseases including cystic fibrosis and chronic obstructive pulmonary disease (COPD) and has proven a valuable therapeutic target. Using human bronchial epithelial cells (hBECs) isolated differentiated at air-liquid interface we demonstrated a reduced function of the CFTR in non-eosinophilic asthma. Characterization of the cell and molecular differences in airway epithelial cells between severe asthma subtypes using single cell RNA-sequencing (scRNAseq) revealed that airway epithelial cells from non-eosinophilic asthma, and in particular neutrophilic asthma patients, fail to differentiate into CFTR-expressing ionocytes compared with eosinophilic asthma or healthy donors. We identified a novel ionocyte transcriptional signature, which was present in both bronchial and tracheal airway epithelial samples indicating conserved anatomical gene regulation. Using protein markers and immunofluorescent quantification loss of ionocytes was confirmed in non-eosinophilic asthma hBECs. Similarly, ioncytes were also diminished in the airways of a murine model of neutrophilic-dominant but not eosinophilic allergen asthma models. Furthermore, treatment of hBECs from healthy donors with a neutrophilic asthma-like inflammatory cytokine mixture, but not IL-13, led to loss of ionocytes primarily due to IFN-{gamma}. Inflammation-induced loss of CFTR-expressing ionocytes in airway cells from non-eosinophilic asthma may represent a key feature of disease pathogenesis and a novel drug target for this difficult-to-treat disease.

cell biology↗

bioRxiv2022

Lung epithelial cell-derived C3 protects against pneumonia-induced lung injury

The complement component C3 is a fundamental plasma protein for host defense. However, recent work has demonstrated the critical importance of local C3 expression in cell survival. Here we analyzed the effects of local versus peripheral sources of C3 expression in a model of bacterial pneumonia. While mice with global C3 deficiency had severe pneumonia-induced lung injury, those deficient in liver-deficient C3 remain protected, comparable to wildtype mice. Human lung transcriptome analysis showed secretory epithelial cells are a major source of C3. Mice with a C3 gene ablation from lung epithelial cells had worse pulmonary injury compared to wild type, despite maintaining normal circulating C3 levels. Finally, in human cellular and mouse pneumonia models, we show that C3 reduces epithelial cell death mediated through the alternative pathway component Factor B. Thus, our findings suggest that a locally-derived C3-Factor B pathway protects the lung mucosal barrier. One Sentence SummaryLung-derived C3 mitigates severe bacterial pneumonia suggesting a novel cytoprotective role at mucosal barrier surfaces independent of circulating C3.

immunology↗

bioRxiv2022

Engineering Rotating Apical-Out Airway Organoid for Assessing Respiratory Cilia Motility

Motile cilia project from the airway apical surface and directly interface with inhaled external environment. Due to cilias nanoscale dimension and high beating frequency, quantitative assessment of their motility remains a sophisticated task. Here we described a robust approach for reproducible engineering of apical-out airway organoid (AOAO) of defined size. Propelled by exterior-facing cilia beating, the mature AOAO exhibited stable rotational motion when surrounded by Matrigel. We developed a computational framework leveraging computer vision algorithms to quantify AOAO rotation and validated its correlation with direct measurement of cilia motility. We further established the feasibility of using AOAO rotation to recapitulate and measure defective cilia motility caused by chemotherapy-induced toxicity and by CCDC39 mutations in cells from primary ciliary dyskinesia patient. We expect our rotating AOAO model and the associated computational pipeline to offer a generalizable framework to expediate modeling of and therapeutic development for genetic and environmental ciliopathies.

bioengineering↗