Faculty Bibliography

Background/Purpose: The skin is recognized as a window into the immunopathogenic mechanisms driving the vast phenotypic spectrum of psoriatic disease.
Method(s): To better decipher the cellular landscape of both healthy and psoriatic skin, we employed spatial transcriptomics (ST), a ground-breaking technology that precisely maps gene expression from histologically-intact tissue sections (Fig. 1A).
Result(s): Findings gleaned from computationally integrating our 23 matched lesional and non-lesional psoriatic and 7 healthy control samples with publicly-available single-cell ribonucleic acid (RNA) sequencing datasets established the ability of ST to recapitulate the tissue architecture of both healthy and inflamed skin (Fig. 1B) and highlighted topographic shifts in the immune cell milieu, from a predominantly perifollicular distribution in steady-state skin to the papillary and upper reticular dermis in psoriatic lesional skin. We also incidentally discovered that ST's ability to ascertain gene expression patterns from intact tissue rendered it particularly conducive to studying the transcriptome of lipid-laden cells such as dermal adipose tissue and sebaceous glands (Fig. 1C), whose expression profiles are typically lost in the process of tissue handling and dissociation for bulk and single-cell RNA seq. Unbiased clustering of pooled healthy and psoriatic samples identified two epidermal clusters and one dermal cluster that were differentially expanded in psoriatic lesional skin (p values <=0.05) (Fig. 1D); pathway analysis of these clusters revealed enrichment of known psoriatic inflammatory pathways (Fig. 1E). Unsupervised classification of skin-limited psoriasis and psoriatic arthritis samples revealed stratification by cutaneous disease severity or Psoriasis Area and Severity Index (PASI) score and not by presence or absence of concomitant systemic/synovial disease (Fig. 1F). Remarkably, this PASI-dependent segregation was also evident in distal, non-lesional samples and was driven by the dermal macrophage and fibroblast cluster and the lymphatic endothelium (Fig. 2A). Inquiry into the mechanistic drivers of this observed stratification yielded enrichment of pathways associated with key T cell and innate immune cell activation, B cells, and metabolic dysfunction (Fig. 2B). Finally, tissue scale computational cartography of gene expression revealed differences in regional enrichment of specific cell types across phenotypic groups, most notably upward extension of fibroblasts to the upper dermis in both lesional and non-lesional samples from mild psoriasis and restriction to the lower dermis in the moderate-to-severe psoriasis samples (Fig. 2C), suggesting that disease severity stratification may be driven by emergent cellular ecosystems in the upper dermis. Fig. 1. (A) Schematic of spatial transcriptomics study workflow. Four mm skin punch biopsies were obtained from healthy volunteers (n=3) and lesional and non-lesional skin from patients with psoriatic disease (n=11). Ten micron-thick sections were then placed on capture areas on the ST microarray slide, each containing molecularly barcoded, spatially encoded spots with a diameter of 50 microns and a center-to-center distance of 100 microns. (B) Side-by-side comparison of a hematoxylin-eosin (H&E) stained section of representative healthy, lesional, and non-lesional skin samples and the corresponding ST plots showed concordance of unbiased gene expression-based clustering with histologic tissue architecture. (C) Pathway analysis of the adipose cluster in healthy skin (cluster 2) confirmed upregulation of lipid-associated processes. Inset: Spots corresponding to the adipose cluster highlighted in yellow. (D) Wilcoxon rank sum test (results displayed as box plots) yielded statistically significant expansion of three clusters in lesional skin compared to both non-lesional and healthy skin-inflamed suprabasal epidermis (cluster 4), epidermis 2 (cluster 7), and inflamed dermis (cluster 10). HC=healthy control, L=lesional psoriatic skin, NL=non-lesional psoriatic skin. (E) Pathways enriched in clusters 4, 7, and 10. (F) Principal component analysis (PCA) plots demonstrating segregation of samples by severity of cutaneous disease in both lesional and non-lesional samples along the first principal component (right) that was not seen in the samples categorized according to presence or absence of arthritis (left). PsA=psoriatic arthritis, PsO=skin-limited psoriasis. Fig. 2. (A) PCA of lesional and non-lesional samples colored by disease severity in spatial clusters 1 (left) and 12 (right) revealed more discrete clustering. (B) Pathways significantly enriched in clusters 1 (left) and 12 (right) showed enrichment of pathways associated with key T cell and innate immune cell activation, B cells, and metabolic dysfunction (highlighted in red). (C) SpaceFold one dimension projection of cell distribution from an independently-generated single-cell RNA seq data set on aggregated ST lesional and non-lesional samples from mild (PASI-low) and moderate-severe (PASI-high) samples. Y-axis represents tissue position, starting with the lower dermis marked as position 0 to suprabasal epidermis marked as position 1. Dashed line represents epidermal-dermal junction, discerned by cell types in the basal epidermal layer (melanocytes and Langerhans cells). Fibroblast signatures (red arrows) were largely relegated to the lower dermis in the PASI-high group, but extended to the upper dermis in the PASI-low group. This striking difference in fibroblast localization was also noted in non-lesional PASI-high vs. PASI-low groups. In addition to fibroblasts, lymphatic, endothelial, myeloid, and T cells signatures (black arrows) were also observed in the upper dermis of lesional PASI-low samples, but were much lower in the dermis of PASI-low non-lesional and all samples in the PASI-high group. Interfollicular epidermis (IFE), hair follicle and infundibulum (HF/IFN), n= number of individual biopsies.
Conclusion(s): Thus, we have been able to successfully leverage ST integrated with independently-generated single-cell RNA seq data to spatially define the emergent cellular ecosystems of healthy and matched psoriatic lesional and non-lesional skin and in so doing, demonstrated the value of ST in unearthing the genetic groundwork at both the site of inflammation and in distal, clinically-uninvolved skin

We identified a Delta-Omicron SARS-CoV-2 recombinant in an unvaccinated, immunosuppressed kidney transplant recipient who had positive COVID-19 tests in December 2021 and February 2022 and was initially treated with Sotrovimab. Viral sequencing in February 2022 revealed a 5' Delta AY.45 portion and a 3' Omicron BA.1 portion with a recombination breakpoint in the spike N-terminal domain, adjacent to the Sotrovimab quaternary binding site. The recombinant virus induced cytopathic effects with characteristics of both Delta (large cells) and Omicron (cell rounding/detachment). Monitoring of immunosuppressed COVID-19 patients treated with antiviral monoclonal antibodies is crucial to detect potential selection of recombinant variants.

Mammalian cells autonomously activate hypoxia-inducible transcription factors (HIFs) to ensure survival in low-oxygen environments. We report here that injury-induced hypoxia is insufficient to trigger HIF1α in damaged epithelium. Instead, multimodal single-cell and spatial transcriptomics analyses and functional studies reveal that retinoic acid-related orphan receptor γt⁺ (RORγt⁺) γδ T cell-derived interleukin-17A (IL-17A) is necessary and sufficient to activate HIF1α. Protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling proximal of IL-17 receptor C (IL-17RC) activates mammalian target of rapamycin (mTOR) and consequently HIF1α. The IL-17A-HIF1α axis drives glycolysis in wound front epithelia. Epithelial-specific loss of IL-17RC, HIF1α, or blockade of glycolysis derails repair. Our findings underscore the coupling of inflammatory, metabolic, and migratory programs to expedite epithelial healing and illuminate the immune cell-derived inputs in cellular adaptation to hypoxic stress during repair.

BACKGROUND:In 2021, Delta became the predominant SARS-CoV-2 variant worldwide. While vaccines have effectively prevented COVID-19 hospitalization and death, vaccine breakthrough infections increasingly occurred. The precise role of clinical and genomic determinants in Delta infections is not known, and whether they contributed to increased rates of breakthrough infections compared to unvaccinated controls. METHODS:We studied SARS-CoV-2 variant distribution, dynamics, and adaptive selection over time in relation to vaccine status, phylogenetic relatedness of viruses, full genome mutation profiles, and associated clinical and demographic parameters. FINDINGS/RESULTS:We show a steep and near-complete replacement of circulating variants with Delta between May and August 2021 in metropolitan New York. We observed an increase of the Delta sublineage AY.25 (14% in vaccinated, 7% in unvaccinated), its spike mutation S112L, and AY.44 (8% in vaccinated, 2% in unvaccinated) with its nsp12 mutation F192V in breakthroughs. Delta infections were associated with younger age and lower hospitalization rates than Alpha. Delta breakthrough infections increased significantly with time since vaccination, and, after adjusting for confounders, they rose at similar rates as in unvaccinated individuals. INTERPRETATION/CONCLUSIONS:We observed a modest adaptation of Delta genomes in breakthrough infections in New York, suggesting an improved genomic framework to support Delta's epidemic growth in times of waning vaccine protection despite limited impact on vaccine escape. FUNDING/BACKGROUND:The study was supported by NYU institutional funds. The NYULH Genome Technology Center is partially supported by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center.

Embryos are diagnosed as mosaic if their chromosomal copy number falls between euploid and aneuploid. The purpose of this study was to investigate the impact of mosaicism on global gene expression. This study included 42 blastocysts that underwent preimplantation genetic testing for aneuploidy (PGT-A) and were donated for IRB approved research. Fourteen blastocysts were diagnosed as mosaic with Next-generation Sequencing (NGS). Three NGS diagnosed euploid embryos, and 25 aneuploid embryos (9 NGS, 14 array Comparative Genomic Hybridization, 2 Single Nucleotide Polymorphism array) were used as comparisons. RNA-sequencing was performed on all of the blastocysts. Differentially expressed genes (DEGs) were calculated using DESeq2/3.5 (R Bioconductor Package) with p < 0.05 considered significantly differentially expressed. Pathway analysis was performed on mosaic embryos using EnrichR with p < 0.05 considered significant. With euploid embryo gene expression used as a control, 12 of 14 mosaic embryos had fewer DEGs compared to aneuploid embryos involving the same chromosome. On principal component analysis (PCA), mosaic embryos mapped separately from aneuploid embryos. Pathways involving cell proliferation, differentiation, and apoptosis were the most disrupted within mosaic embryos. Mosaic embryos have decreased disruption of global gene expression compared to aneuploid embryos. This study was limited by the small sample size, lack of replicate samples for each mosaic abnormality, and use of multiple different PGT-A platforms for the diagnosis of aneuploid embryos.

Breast cancer represents the most common cancer diagnosis among World Trade Center (WTC)-exposed community members, residents, and cleanup workers enrolled in the WTC Environmental Health Center (WTC EHC). The primary aims of this study were (1) to compare blood DNA methylation profiles of WTC-exposed community members with breast cancer and WTC-unexposed pre-diagnostic breast cancer blood samples, and (2) to compare the DNA methylation differences among the WTC EHC breast cancer cases and WTC-exposed cancer-free controls. Gene pathway enrichment analyses were further conducted. There were significant differences in DNA methylation between WTC-exposed breast cancer cases and unexposed prediagnostic breast cancer cases. The top differentially methylated genes were Intraflagellar Transport 74 (IFT74), WD repeat-containing protein 90 (WDR90), and Oncomodulin (OCM), which are commonly upregulated in tumors. Probes associated with established tumor suppressor genes (ATM, BRCA1, PALB2, and TP53) were hypermethylated among WTC-exposed breast cancer cases compared to the unexposed group. When comparing WTC EHC breast cancer cases vs. cancer-free controls, there appeared to be global hypomethylation among WTC-exposed breast cancer cases compared to exposed controls. Functional pathway analysis revealed enrichment of several gene pathways in WTC-exposed breast cancer cases including endocytosis, proteoglycans in cancer, regulation of actin cytoskeleton, axon guidance, focal adhesion, calcium signaling, cGMP-PKG signaling, mTOR, Hippo, and oxytocin signaling. The results suggest potential epigenetic links between WTC exposure and breast cancer in local community members enrolled in the WTC EHC program.

Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. "Drug-tolerant persisters" (DTPs), a sub-population of cancer cells that survive via reversible, non-genetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKIs) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single cell RNA-sequencing reveal that HER2+ breast cancer cells cycle stochastically through a "pre-DTP" state, characterized by a G0-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting show that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation.

Of 379 severe acute respiratory syndrome coronavirus 2 samples collected in New York, USA, we detected 86 Omicron variant sequences containing Delta variant mutation P681R. Probable explanations were co-infection with 2 viruses or contamination/amplification artifact. Repeated library preparation with fewer cycles showed the P681R calls were artifactual. Unusual mutations should be interpreted with caution.