@ BRIC-inStem
Research
We focus on conserved principles by which epithelial identity in barrier tissues is shaped by developmental cues and subsequently reinforced through interactions with commensal microbiota, systemic signals such as steroid hormones, and ongoing immune-epithelial crosstalk.
A central interest of the lab is understanding how epithelial differentiation across continuous tissue surfaces, such as the genitourinary tract, gives rise to spatially organized resident immune cell niches. We examine how gradients of epithelial gene expression, particularly chemokines and other immune-modulatory factors pattern the localization, density, and functional specialization of tissue-resident immune cells at barrier surfaces exposed to the external environment. To address these questions, we combine mouse models and organotypic culture systems with spatial and single-cell transcriptomic approaches to resolve epithelial and immune heterogeneity at high resolution. We investigate how shared developmental origins can diverge into distinct barrier states along anatomical axes, providing a tractable framework to study how epithelial transcriptional programs coordinate local immune architecture.
Making and Breaking Epithelial Barriers
Epithelial cells form specialized linings that form protective surfaces in the body. Epithelial cells stick together with adhesion proteins to form tight and selectively permeable linings that form a physical barrier against infection. Epithelial barriers can be considered as the walls of a fort that protect vulnerable occupants inside the walls of the fort. Epithelial barriers form during fetal development and are gradually patterned along a proximal-distal axis to express high levels of defensive proteins at vulnerable sites like external openings.
Much like sentinels on the ramparts of a fort, resident immune cells patrol and survey the epithelial barrier to detect any potential threats. The organization of immune cells along the epithelial barrier is guided by signaling gradients from epithelial cells that are in turn influenced by developmentally patterned transcription factor expression, microbial cues and hormonal input.
In response to pathogens, the epithelial barrier and resident immune cells get activated to defend the body and recruit reinforcements from the circulating immune cell pool. Damage to epithelial cells is healed by tissue resident stem cells that respond to immune and stromal derived growth cues to repair the barrier.
Our recent work shows that developmentally encoded gene expression patterns in the urethra are reinforced postnatally to form proximal-distal chemokine gradients in the adult urethra. This patterning may help attract macrophages via secreted chemokines to vulnerable sites like the external urethral opening where expression of the chemokines is highest.
We found that considerable changes happen to the epithelial lining of the urethra right after birth. The expression of mucosal chemokine genes in the epithelial lining increases in the postnatal period and correlates with higher infiltration of resident macrophages into the epithelial lining. We found that macrophages embedded in the epithelial lining represent a transcriptionally distinct, activated macrophage subtype (Mac-Activated) compared to the homeostatic macrophages with scavenging capacity located in the stromal compartment (Mac-Endocytic).
Ongoing work in the lab investigates critical cell signaling pathways that maintain epithelial integrity and immune homeostasis. Loss of activity of these pathways leads to defective barriers and heightened inflammation. In the context of urinary tract infections (UTIs), we investigate how alterations to the epithelial and immune compartment upon infection are reversed to homeostatic levels when infection clears. We investigate long-term remodeling of the epithelial and immune compartments after multiple infectious insults that would occur in patients with recurrent UTIs.
Approaches
Epithelia in the body are diverse in terms of cellular architecture, organization and function. We study different epithelial linings in the body to identify conserved and distinct mechanisms that maintain barrier integrity, immune homeostasis and defensive capacities.
Female Reproductive Tract
Oesophagus
Lower Urinary Tract
 We investigate how defense mechanisms are patterned along the proximal-distal axis of the lower urinary tract to protect against UTIs. |  We investigate how hormonal input and cues from the commensal microbiome establish epithelial gene expression patterns and resident immune cell organization in the female reproductive tract. |
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 We investigate signaling pathways maintaining epithelial barrier integrity, differentiation and immune homeostasis in the oesophagus. |
We use mice to model development and postnatal maturation of epithelial linings as well as responses to infection. Using transgenic mice, we follow particular cell populations over time and disrupt specific genes to understand their role in maintaining epithelial barrier function.
We generate epithelial organoids from tissue resident stem cells or induced pluripotent stem cells to investigate epithelial differentiation. We use organoids to study epithelial-immune interactions and model infection.
We perform tissue profiling using single cell RNA-sequencing, spatial transcriptomics and multiplex immunostaining/in situ hybridization for assessing cellular heterogeneity and responses to inflammation and barrier disruption.
Early epithelial patterning and establishment of immune defenses in the lower urinary tract
Epithelial barriers represent the first line of defense against an invasive pathogenic attack. Formation of epithelial barriers requires patterning of contiguous epithelial tissue during development to generate specialized linings of organs. My first goal when establishing my program at inStem was to understand how gene expression in the urethra and bladder are patterned from the urogenital sinus and how immune defenses are established in the urethra during fetal development. In this study, we provide a direct comparison of gene expression in the urethra and bladder during development, along with early transcription factors that are restricted to the bladder or urethra. We defined epithelial and mesenchymal gene signatures that are specific to the developing bladder or urethra. Previous studies have addressed bladder development in isolation from the urethra. By providing a direct comparison of gene expression in the epithelial and mesenchymal compartments of the lower urinary tract, we identified novel marker genes, transcription factors and signaling pathway genes that are spatially restricted to the bladder or urethra. Additionally, we identified a novel signature of chemokine gene expression in the urethral epithelium which correlated with the appearance of epithelial-associated macrophages. Lastly, our study revealed that the urethra shows sexually dimorphic gene expression in the epithelial and mesenchymal compartments while the bladder does not have the same degree of sexual dimorphism.
Identification of novel defense mechanisms in the female urethra
Urinary pathogens have to ascend the urethra to reach the bladder. The protective mechanisms in the urethra and how these are established are not known. We generated the first cellular atlas of the adult female mouse urethra revealing transcriptionally distinct basal, intermediate and luminal cells in the epithelial lining. Our work reveals the diversity of resident immune cell populations in the female urethra and identifies two spatially separated and transcriptionally distinct macrophage populations. One population of epithelial-associated macrophages displays a large cell perimeter and sends out dendrites in between epithelial cells. The other macrophage population resides in the stromal compartment and expresses gene signatures for scavenging, vascular development and tissue homeostasis. We also identify two secretory epithelial cell types in the adult female mouse urethra which express chemoattractant genes. Our work additionally uncovered novel urethral secretory glands which connect to the trunk of the urethra through short ducts. The urethral glands express antimicrobial genes and transporters for the acidification of lumen contents. Expression of chemoattractant genes in the urethral lining increases as we move closer to the outside of the body and correlates with the density of epithelial-associated macrophages which also increases towards the urethral opening. This study sheds light on an understudied mucosal barrier that is at the frontline of the response to urinary pathogens, identifying novel macrophage populations and epithelial gene signatures that can influence immunity.
Tissue-derived urethral organoids recapitulate epithelial stratification and highlight proximal-distal differences in stem cell activity
We published detailed methods to study the epithelial lining of the female mouse urethra and make 3D urethral organoids. We described methods for dissection of the full length female mouse urethra and isolation of the epithelial lining by enzymatic and mechanical dissociation. With the isolated epithelial lining, we performed wholemount immunostaining to observe immune cells embedded in the epithelial lining and also performed flow cytometry on the isolated epithelial compartment. The viable urethral epithelial cells from this protocol formed stratified 3D urethral organoids which express markers of urethral epithelium and are formed with higher efficiency from cells from the distal urethra, suggesting proximal-distal differences in cell composition and stemness. Urethral organoids can serve as useful models to study epithelial stratification and infection in vitro