Faculty Bibliography

Infections and inflammation are profoundly influenced by the extracellular matrix (ECM), but their molecular underpinnings are ill defined. Here, we demonstrate that lumican, an ECM protein normally associated with collagens, is elevated in sepsis patients' blood, while lumican-null mice resolve polymicrobial sepsis poorly, with reduced bacterial clearance and greater body weight loss. Secreted by activated fibroblasts, lumican promotes Toll-like receptor (TLR) 4 response to bacterial lipopolysaccharides (LPS) but restricts nucleic acid-specific TLR9 in macrophages and dendritic cells. The underlying mechanism involves lumican attachment to the common TLR coreceptor CD14 and caveolin 1 (Cav1) in lipid rafts on immune cell surfaces via two epitopes, which may be cryptic in collagen-associated lumican. The Cav1 binding epitope alone is sufficient for cell surface enrichment of Cav1, while both are required for lumican to increase cell surface TLR4, CD14, and proinflammatory cytokines in response to LPS. Endocytosed lumican colocalizes with TLR4 and LPS and promotes endosomal induction of type I interferons. Lumican-null macrophages show elevated TLR9 in signal-permissive endolysosomes and increased response, while wild types show lumican colocalization with CpG DNA but not TLR9, consistent with a ligand sequestering, restrictive role for lumican in TLR9 signaling. In vitro, lumican competes with CD14 to bind CpG DNA; biglycan, a lumican paralog, also binds CpG DNA and suppresses TLR9 response. Thus, lumican and other ECM proteins, synthesized de novo or released from collagen association during ECM remodeling, may be internalized by immune cells to regulate their transcriptional programs and effector responses that may be harnessed in future therapeutics.

In this review we highlight emerging immune regulatory functions of lumican, keratocan, fibromodulin, biglycan and decorin, which are members of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). These SLRPs have been studied extensively as collagen-fibril regulatory structural components of the skin, cornea, bone and cartilage in homeostasis. However, SLRPs released from a remodeling ECM, or synthesized by activated fibroblasts and immune cells contribute to an ECM-free pool in tissues and circulation, that may have a significant, but poorly understood foot print in inflammation and disease. Their molecular interactions and the signaling networks they influence also require investigations. Here we present studies on the leucine-rich repeat (LRR) motifs of SLRP core proteins, their evolutionary and functional relationships with other LRR pathogen recognition receptors, such as the toll-like receptors (TLRs) to bring some molecular clarity in the immune regulatory functions of SLRPs. We discuss molecular interactions of fragments and intact SLRPs, and how some of these interactions are likely modulated by glycosaminoglycan side chains. We integrate findings on molecular interactions of these SLRPs together with what is known about their presence in circulation and lymph nodes (LN), which are important sites of immune cell regulation. Recent bulk and single cell RNA sequencing studies have identified subsets of stromal reticular cells that express these SLRPs within LNs. An understanding of the cellular source, molecular interactions and signaling consequences will lead to a fundamental understanding of how SLRPs modulate immune responses, and to therapeutic tools based on these SLRPs in the future.

Lumican is an extracellular matrix proteoglycan known to regulate toll-like receptor (TLR) signaling in innate immune cells. In experimental settings, lumican suppresses TLR9 signaling by binding to and sequestering its synthetic ligand, CpG-DNA, in non-signal permissive endosomes. However, the molecular details of lumican interactions with CpG-DNA are obscure. Here, the 3-D structure of the 22 base-long CpG-DNA (CpG ODN_2395) bound to lumican or TLR9 were modeled using homology modeling and docking methods. Some of the TLR9-CpG ODN_2395 features predicted by our model are consistent with the previously reported TLR9-CpG DNA crystal structure, substantiating our current analysis. Our modeling indicated a smaller buried surface area for lumican-CpG ODN_2395 (1803 Ų) compared to that of TLR9-CpG ODN_2395 (2094 Ų), implying a potentially lower binding strength for lumican and CpG-DNA than TLR9 and CpG-DNA. The docking analysis identified 32 amino acids in lumican LRR1-11 interacting with CpG ODN_2395, primarily through hydrogen bonding, salt-bridges, and hydrophobic interactions. Our study provides molecular insights into lumican and CpG-DNA interactions that may lead to molecular targets for modulating TLR9-mediated inflammation and autoimmunity.

The cornea is a protective and refractive barrier in the eye crucial for vision. Understanding the human cornea in health, disease, and cell-based treatments can be greatly advanced with cornea organoids developed in culture from induced pluripotent stem cells. While a limited number of studies have investigated the single-cell transcriptomic composition of the human cornea, its organoids have not been examined similarly. Here, we elucidated the transcriptomic cell fate map of 4-month-old human cornea organoids and human donor corneas. The organoids harbor cell clusters that resemble cells of the corneal epithelium, stroma, and endothelium, with subpopulations that capture signatures of early developmental states. Unlike the adult cornea where the largest cell population is stromal, the organoids contain large proportions of epithelial and endothelial-like cells. These corneal organoids offer a 3D model to study corneal diseases and integrated responses of different cell types.

Metazoans have evolved to produce various types of extracellular matrix (ECM) that provide structural support, cell adhesion, cell-cell communication, and regulated exposure to external cues. Epithelial cells produce and adhere to a specialized sheet-like ECM, the basement membrane, that is critical for cellular homeostasis and tissue integrity. Mesenchymal cells, such as chondrocytes in cartilaginous tissues and keratocytes in the corneal stroma, produce a pericellular matrix that presents optimal levels of growth factors, cytokines, chemokines, and nutrients to the cell and regulates mechanosensory signals through specific cytoskeletal and cell surface receptor interactions. Here, we discuss laminins, collagen types IV and VII, and perlecan, which are major components of these two types of ECM. We examine genetic defects in these components that cause basement membrane pathologies such as epidermolysis bullosa, Alport syndrome, rare pericellular matrix-related chondrodysplasias, and corneal keratoconus and discuss recent advances in cell and gene therapies being developed for some of these disorders. Expected final online publication date for the Annual Review of Genomics and Human Genetics, Volume 23 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Keratoconus (KC) is a degenerative disease associated with cell and extracellular matrix (ECM) loss that causes gradual thinning and steepening of the cornea and loss of vision. Collagen cross linking with ultraviolet light treatment can strengthen the ECM and delay weakening of the cornea, but severe cases require corneal transplantation. KC is multifactorial and multigenic, but its pathophysiology is still an enigma. Multiple approaches are being pursued to elucidate the molecular changes that underlie the corneal phenotype to identify relevant genes for tailored candidate searches and to develop potential biomarkers and targets for therapeutic interventions. Recent proteomic and transcriptomic studies suggest dysregulations in oxidative stress, NRF2-regulated antioxidant programs, WNT-signaling, TGF-β, ECM and matrix metalloproteinases. This review aims to provide a broad update on the transcriptomic and proteomic studies of KC with a focus on findings that relate to oxidative stress, and dysregulations in cellular and extracellular matrix functions.

Purpose : The hallmark of keratoconus (KC) is biomechanical failure of the cornea, with fragmentation of Bowman's layer (BL) contributing to reduced corneal strength. Proteomic studies of collagen I in the KC corneal epithelium have shown underexpression of this major structural component of the extracellular matrix. Collagen I spatially localizes to BL and is produced in part by corneal epithelium. Wnt signaling, which regulates extracellular matrix production, has been shown to be dysregulated in KC, and our transcriptomic study of progressive KC corneal epithelium demonstrates underexpression of WNT10A mRNA. However, it is unclear whether WNT10A regulates collagen I expression in the corneal epithelium. Methods : RNA-sequencing was performed on corneal epithelium samples of five progressive KC and five myopic control eyes. WNT10A underexpression was validated using TaqMan qPCR on 31 additional independent samples; protein level validation with Western blot analysis. Immunohistochemistry was performed on tissue microarrays containing cores from over 100 KC and control cases. Additionally, WNT10A was overexpressed in vitro in immortalized corneal epithelial cells. Results : WNT10A was underexpressed in KC corneal epithelium as compared to myopic controls (transcript ratio KC/control=0.59, p=0.02 per RNA-sequencing study; transcript ratio=0.66, p=0.03 per qPCR; protein ratio=0.07, p=0.06 per Western blot). Immunohistochemical analysis demonstrated WNT10A absence or marked decrease in BL of KC corneas (p<0.0001). Finally, WNT10A positively regulated COL1A1 expression in corneal epithelial cells (protein ratio=11, p=0.02). Conclusions : Though long suggested historically, our results provide for the first time a potential molecular mechanism supporting the hypothesis that alterations of the epithelium contribute to stromal pathogenesis in KC. Specifically, we find that WNT10A promotes COL1A1 expression, and hypothesize that when WNT10A levels are low, reduced deposition of collagen I in BL may compromise biomechanical strength and potentiate breaks typical of KC. These studies also suggest that the Wnt pathway represents a therapeutic target, as its manipulation in the corneal epithelium may induce collagen I production and increase tensile strength in KC

Purpose : To establish a well-defined 3D corneal organoid model that will be useful in studying ocular surface diseases, their genetic modeling and screening of pharmaceutical drugs. We performed single-cell RNA sequencing (scRNA-seq) of a human donor cornea and a cornea organoid developed in culture from induced pluripotent stem cells (iPSC) to elucidate corneal cell populations represented in the organoid. Methods : One cornea organoid (7 month old), and a healthy donor (41 year old) cornea (Lions Eye Institute for Transplant and Research, FL) were digested for 5 h with Collagenase type I (2 mg/ml) in complete DMEM-F12 containing 5% FBS followed by Accutase treatment for 20 min, washed and re-suspended in PBS with 0.04% BSA. Live cells were counted by trypan-blue exclusion in a Countess II automated cell counter. For scRNA-seq, the cells were captured and libraries generated using Chromium Single Cell 3' Library & Gel Bead Kit v2 (10x Genomics). Libraries were run on an Illumina HiSeq 4000 as 150-bp paired-end reads. The Cell Ranger Single-Cell Software Suite v3.01 was used to perform sample demultiplexing, barcode processing and single-cell 3' gene counting. Quality control filtering was applied to remove any cells with fewer than 1000 reads or greater that 15% mitochondrial reads. The 10x Chromium Single Cell 3' Library & Gel Bead Kit v2 was used to capture cells from the donor cornea (10,000 cells) and the organoid (2792 cells). The Loupe Browser 4.2.0 was used to analyze and visualize the data. Results : We detected 11 and 18 cell clusters in the cornea organoid and donor cornea, respectively. Among these, the cornea organoid revealed clusters of epithelial cells expressing MUC1 and MUC16, a small set of TP63 cells and a stromal cell cluster expressing COL1A1, COL5A, and a small subset of these expressing LUM and KERA. Unlike the cornea, the organoid also contained cells expressing KRT13 and MUC4, indicative of cornea atypical epithelial differentiation. We also found the expression of SARS-CoV2 receptors (ACE2 and TMPRSS2) in the organoid and the donor cornea. Conclusions : The cornea organoid displayed considerable overlap with the cornea with respect to stromal cell and some epithelial cell markers. However, the organoid also harbored a cluster indicative of dermal and conjunctival differentiation

Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and, in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2, and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared to 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

Purpose/UNASSIGNED:To identify global gene expression changes in the corneal epithelium of keratoconus (KC) patients compared to non-KC myopic controls. Methods/UNASSIGNED:RNA-sequencing was performed on corneal epithelium samples of five progressive KC and five myopic control patients. Selected results were validated using TaqMan quantitative PCR (qPCR) on 31 additional independent samples, and protein level validation was conducted using western blot analysis on a subset. Immunohistochemistry was performed on tissue microarrays containing cores from over 100 KC and control cases. WNT10A transcript levels in corneal epithelium were correlated with tomographic indicators of KC disease severity in 15 eyes. Additionally, WNT10A was overexpressed in vitro in immortalized corneal epithelial cells. Results/UNASSIGNED:WNT10A was found to be underexpressed in KC epithelium at the transcript (ratio KC/control = 0.59, P = 0.02 per RNA-sequencing study; ratio = 0.66, P = 0.03 per qPCR) and protein (ratio = 0.07, P = 0.06) levels. Immunohistochemical analysis also indicated WNT10A protein was decreased in Bowman's layer of KC patients. In contrast, WNT10A transcript level positively correlated with increased keratometry (Kmax ρ = 0.57, P = 0.02). Finally, WNT10A positively regulated COL1A1 expression in corneal epithelial cells. Conclusions/UNASSIGNED:A specific Wnt ligand, WNT10A, is reduced at the mRNA and protein level in KC epithelium and Bowman's layer. This ligand positively regulates collagen type I expression in corneal epithelial cells. The results suggest that WNT10A expression in the corneal epithelium may play a role in progressive KC.