Faculty Bibliography

BACKGROUND:The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial (HAE) cultures at air-liquid interface are a physiologically relevant in vitro model of this heterogeneous tissue and have enabled numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining the capacity for differentiation to HAE. However, gene expression and innate immune function in BCi-NS1.1-derived versus primary-derived HAE cultures have not been fully characterized. METHODS:BCi-NS1.1-derived HAE cultures (n = 3 independent differentiations) and primary-derived HAE cultures (n = 3 distinct donors) were characterized by immunofluorescence and single cell RNA-Seq (scRNA-Seq). Innate immune functions were evaluated in response to interferon stimulation and to infection with viral and bacterial respiratory pathogens. RESULTS:We confirm at high resolution that BCi-NS1.1- and primary-derived HAE cultures are largely similar in morphology, cell type composition, and overall gene expression patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1-derived HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus. CONCLUSIONS:Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

Stem cell quiescence, proliferation and differentiation are controlled by interactions with niche cells and a specialized extracellular matrix called basement membrane (BM). Direct interactions with adjacent BM are known to regulate stem cell quiescence; however, it is less clear how niche BM relays signals to stem cells that it does not contact. Here, we examine how niche BM regulates Caenorhabditis elegans primordial germ cells (PGCs). BM regulates PGC quiescence even though PGCs are enwrapped by somatic niche cells and do not contact the BM; this can be demonstrated by depleting laminin, which causes normally quiescent embryonic PGCs to proliferate. We show that following laminin depletion, niche cells relay proliferation-inducing signals from the gonadal BM to PGCs via integrin receptors. Disrupting the BM proteoglycan perlecan blocks PGC proliferation when laminin is depleted, indicating that laminin functions to inhibit a proliferation-inducing signal originating from perlecan. Reducing perlecan levels in fed larvae hampers germline growth, suggesting that BM signals regulate germ cell proliferation under physiological conditions. Our results reveal how BM signals can regulate stem cell quiescence indirectly, by activating niche cell integrin receptors.

BACKGROUND:Tau protein serves a pro-inflammatory function in neuroinflammation. However, the role of tau in other inflammatory disorders such as rheumatoid arthritis (RA) is less explored. This study is to investigate the role of endogenous tau and the potential mechanisms in the pathogenesis of inflammatory arthritis. METHODS:We established collagen-induced arthritis (CIA) model in wild-type and Tau-/- mice to compare the clinical score and arthritis incidence. Micro-CT analysis was used to evaluate bone erosion of ankle joints. Histological analysis was performed to assess inflammatory cell infiltration, cartilage damage, and osteoclast activity in the ankle joints. Serum levels of pro-inflammatory cytokines were measured by ELISA. The expression levels of macrophage markers were determined by immunohistochemistry staining and quantitative real-time PCR. RESULTS:Tau expression was upregulated in joints under inflammatory condition. Tau deletion in mice exhibited milder inflammation and protected against the progression of CIA, evidenced by reduced serum levels of pro-inflammatory cytokines and attenuated bone loss, inflammatory cell infiltration, cartilage damage, and osteoclast activity in the ankle joints. Furthermore, tau deficiency led to the inhibition of classically activated type 1 (M1) macrophage polarization in the synovium. CONCLUSION:Tau is a previously unrecognized critical regulator in the pathogenesis of RA and may provide a potential therapeutic target for autoimmune and inflammatory joint diseases.

Histone H2A lysine 119 (H2AK119Ub) is monoubiquitinated by Polycomb repressive complex 1 and deubiquitinated by Polycomb repressive deubiquitinase complex (PR-DUB). PR-DUB cleaves H2AK119Ub to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. The PR-DUB subunits (BAP1 and ASXL1) are among the most frequently mutated epigenetic factors in human cancers. How PR-DUB establishes specificity for H2AK119Ub over other nucleosomal ubiquitination sites and how disease-associated mutations of the enzyme affect activity are unclear. Here, we determine a cryo-EM structure of human BAP1 and the ASXL1 DEUBAD in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for restructuring the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing insight into understanding cancer etiology.

Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

Chikungunya virus (CHIKV) infection has been associated with severe cardiac manifestations, yet, how CHIKV infection leads to heart disease remains unknown. Here, we leveraged both mouse models and human primary cardiac cells to define the mechanisms of CHIKV heart infection. Using an immunocompetent mouse model of CHIKV infection as well as human primary cardiac cells, we demonstrate that CHIKV directly infects and actively replicates in cardiac fibroblasts. In immunocompetent mice, CHIKV is cleared from cardiac tissue without significant damage through the induction of a local type I interferon response from both infected and non-infected cardiac cells. Using mice deficient in major innate immunity signaling components, we found that signaling through the mitochondrial antiviral-signaling protein (MAVS) is required for viral clearance from the heart. In the absence of MAVS signaling, persistent infection leads to focal myocarditis and vasculitis of the large vessels attached to the base of the heart. Large vessel vasculitis was observed for up to 60 days post infection, suggesting CHIKV can lead to vascular inflammation and potential long-lasting cardiovascular complications. This study provides a model of CHIKV cardiac infection and mechanistic insight into CHIKV-induced heart disease, underscoring the importance of monitoring cardiac function in patients with CHIKV infections.

To improve the understanding of chemo-refractory high-grade serous ovarian cancers (HGSOCs), we characterized the proteogenomic landscape of 242 (refractory and sensitive) HGSOCs, representing one discovery and two validation cohorts across two biospecimen types (formalin-fixed paraffin-embedded and frozen). We identified a 64-protein signature that predicts with high specificity a subset of HGSOCs refractory to initial platinum-based therapy and is validated in two independent patient cohorts. We detected significant association between lack of Ch17 loss of heterozygosity (LOH) and chemo-refractoriness. Based on pathway protein expression, we identified 5 clusters of HGSOC, which validated across two independent patient cohorts and patient-derived xenograft (PDX) models. These clusters may represent different mechanisms of refractoriness and implicate putative therapeutic vulnerabilities.

Magnetic Resonance Imaging (MRI) resolution continues to improve, making it important to understand the cellular basis for different MRI contrast mechanisms. Manganese-enhanced MRI (MEMRI) produces layer-specific contrast throughout the brain enabling in vivo visualization of cellular cytoarchitecture, particularly in the cerebellum. Due to the unique geometry of the cerebellum, especially near the midline, 2D MEMRI images can be acquired from a relatively thick slice by averaging through areas of uniform morphology and cytoarchitecture to produce very high-resolution visualization of sagittal planes. In such images, MEMRI hyperintensity is uniform in thickness throughout the anterior-posterior axis of sagittal sections and is centrally located in the cerebellar cortex. These signal features suggested that the Purkinje cell layer, which houses the cell bodies of the Purkinje cells and the Bergmann glia, is the source of hyperintensity. Despite this circumstantial evidence, the cellular source of MRI contrast has been difficult to define. In this study, we quantified the effects of selective ablation of Purkinje cells or Bergmann glia on cerebellar MEMRI signal to determine whether signal could be assigned to one cell type. We found that the Purkinje cells, not the Bergmann glia, are the primary of source of the enhancement in the Purkinje cell layer. This cell-ablation strategy should be useful for determining the cell specificity of other MRI contrast mechanisms.

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host^1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis^1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids^4-7 and cholesterol^1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.

PURPOSE/OBJECTIVE:Unlike other cells in the body, in sperm, telomere length (TL) increases with age. TL can regulate nearby genes, and the subtelomeric region is rich in retrotransposons. We hypothesized that age-related telomere lengthening in sperm might suppress Long Interspersed Element 1 (LINE-1/L1), the only competent retrotransposon in humans. METHODS:We measured L1 copy number (L1-CN) and sperm telomere length (STL) from young and older men to evaluate the relationship between age, TL and L1-CN. We also evaluated L1-CN and TL in individual sperm to determine whether these variables influence sperm morphology. STL was assayed by Multiplex quantitative polymerase chain reaction method (mmqPCR) and L1-CN by Quantitative polymerase chain reaction (qPCR). RESULTS:We found that STL increased, and L1-CN decreased significantly with paternal age. STL in normal single sperm was significantly higher than in abnormal sperm. L1-CN did not differ between normal and abnormal sperm. Furthermore, morphologically normal sperm have longer telomeres than abnormal sperm. CONCLUSIONS:Elongation of telomeres in the male germline could repress retrotransposition, which tends to increase with cellular aging. More studies in larger cohorts across a wide age span are needed to confirm our conclusions and explore their biological and clinical significance.