Faculty Bibliography

OBJECTIVE:Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID-19 patients. Given that platelets are key regulators of thrombosis, inflammation, and immunity they represent prime candidates as mediators of COVID-19-associated pathogenesis. The objective of this study was to understand the contribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the platelet phenotype via phenotypic (activation, aggregation) and transcriptomic characterization. APPROACH AND RESULTS/UNASSIGNED:In a cohort of 3915 hospitalized COVID-19 patients, we analyzed blood platelet indices collected at hospital admission. Following adjustment for demographics, clinical risk factors, medication, and biomarkers of inflammation and thrombosis, we find platelet count, size, and immaturity are associated with increased critical illness and all-cause mortality. Bone marrow, lung tissue, and blood from COVID-19 patients revealed the presence of SARS-CoV-2 virions in megakaryocytes and platelets. Characterization of COVID-19 platelets found them to be hyperreactive (increased aggregation, and expression of P-selectin and CD40) and to have a distinct transcriptomic profile characteristic of prothrombotic large and immature platelets. In vitro mechanistic studies highlight that the interaction of SARS-CoV-2 with megakaryocytes alters the platelet transcriptome, and its effects are distinct from the coronavirus responsible for the common cold (CoV-OC43). CONCLUSIONS:Platelet count, size, and maturity associate with increased critical illness and all-cause mortality among hospitalized COVID-19 patients. Profiling tissues and blood from COVID-19 patients revealed that SARS-CoV-2 virions enter megakaryocytes and platelets and associate with alterations to the platelet transcriptome and activation profile.

Cutaneous T cell lymphoma (CTCL) is a heterogeneous group of mature T cell neoplasms characterized by the accumulation of clonal malignant CD4+ T cells in the skin. The most common variant of CTCL, Mycosis Fungoides, is confined to the skin in early stages but can be accompanied by extracutaneous dissemination of malignant T cells to the blood and lymph nodes in advanced stages of disease. Sézary Syndrome, a leukemic form of disease is characterized by significant blood involvement. Little is known about the transcriptional and genomic relationship between skin and blood residing malignant T cells in CTCL. To identify and interrogate malignant clones in matched skin and blood from leukemic MF and SS patients, we combine T cell receptor clonotyping, with quantification of gene expression and cell surface markers at the single cell level. Our data reveals clonal evolution at a transcriptional and genetic level within the malignant populations of individual patients. We highlight highly consistent transcriptional signatures delineating skin-derived and blood-derived malignant T cells. Analysis of these two populations suggests that environmental cues, along with genetic aberrations, contribute to transcriptional profiles of malignant T cells. Our findings indicate that the skin microenvironment in CTCL promotes a transcriptional response supporting rapid malignant expansion, as opposed to the quiescent state observed in the blood, potentially influencing efficacy of therapies. These results provide insight into tissue-specific characteristics of cancerous cells and underscore the need to address the patients' individual malignant profiles at the time of therapy to eliminate all sub-clones.

Background/Purpose: Systemic Lupus Erythematosus (SLE) is a complex chronic heterogeneous autoimmune disease, which increases the risk of atherothrombosis. In addition to their well described role in thrombosis and hemostasis, platelets are key mediators of inflammation and have immune effector cell properties. This study was initiated to investigate the role of platelet associated Lectin Galactoside-binding Soluble 3 Binding Protein (LGALS3BP), which binds to macrophage-associated lectin Mac-2, as a mediator of inflammation in SLE and potential biomarker associated with clinical phenotypes.
Method(s): RNA transcriptome analysis was performed on platelets isolated from 51 patients with SLE (not taking aspirin or anticoagulants) and 18 age, sex and race/ethnicity matched controls. LGALS3BP protein expression was determined in platelet releasates by ELISA and western blot analysis. Gene and protein expression of LGALS3BP in Megakaryocyte cell line (MEG-01) was investigated upon stimulation with IFN-alpha. Correlations between circulating serum LGALS3BP and LGALS3BP platelet mRNA and releasates were assessed. Subsequently, correlation analysis between clinical features of SLE and circulating serum LGLAS3BP was performed. Finally, the effects of platelets and LGALS3BP on macrophage inflammatory response were studied in vitro.
Result(s): Platelet transcriptome analysis revealed that LGALS3BP was one of the most differentially expressed transcripts in SLE versus matched-healthy controls (Fold change, 3.9, adjusted P-value = 2.5 x 10^-11) (Figure1A). Consistently, LGALS3BP in platelet releasates was significantly higher in 40 patients with SLE than 20 controls (p = 0.002) (Figure1B). Platelet LGALS3BP gene and protein expression were highly correlated with circulating LGALRS3BP in serum (r² = 0.370, p = 0.003 and r² = 0.689, p < 0.0001 respectively) (Figure1E and F). LGALS3BP measured in serum of 115 patients with SLE correlated with the SELENA SLEDAI hybrid disease activity index (r²= 0.322, p = 0.0005) (Figure1G). In particular, higher serum LGALS3BP levels were observed in SLE patients with lupus nephritis compared to those with SLE and inactive disease (P=0.0001) (Figure1H). In longitudinal analysis of 22 patients without proteinuria at baseline who went on to develop proteinuria over time, circulating plasma LGALS3BP tracked with flares of nephritis (p=0.06). In vitro, IFN-alpha induced the expression and production of LGALS3BP in MEG-01 cells in a dose dependent manner (Figure2A, B and C), which was completely inhibited by IFN-alpha neutralizing antibody (Figure2D, E and F). Recombinant LGALS3BP (Figure 3A and B) and Platelet releasates from SLE (Figure 3C) induced the production of pro-inflammatory cytokines such as IL-8 (p=0.04) and IL-6 (p=0.073) by macrophages.
Conclusion(s): These data show that platelets isolated from patients with SLE highly express and secrete LGALS3BP which induces a proinflammatory macrophage and is associated with SLE disease clinical phenotype. LGALS3BP may contribute to pathogenesis and serve as a novel biomarker of SLE disease activity

Background/Purpose: The clinical heterogeneity of SLE with its complex pathogenesis remains challenging as we strive to provide optimal management. The contribution of platelets to endovascular homeostasis, inflammation and immune regulation highlights their potential importance in SLE. Prior work from our group showed that the Fcgamma receptor type IIa (FcgammaRIIa)-R/H131 biallelic polymorphism is associated with increased platelet activity and cardiovascular risk in SLE. The study was initiated to investigate the platelet transcriptome in patients with SLE and evaluate its association across FcgammaRIIa genotypes and distinct clinical features.
Method(s): RNA-sequencing was done on platelets isolated from 51 patients fulfilling criteria for the classification of SLE based on recent EULAR/ACR definitions, and 18 healthy controls matched on age, sex, and race. Unsupervised clustering, differential gene expression, and gene set enrichment analysis (GSEA) were used to analyze differences between SLE patients and controls, and SLE subpopulations, based on SELENA SLEDAI Hybrid disease activity, specific organ manifestations, and FcgammaRIIa genotype. Weighted Gene Correlation Network Analysis (WGCNA) was performed to create a modular transcriptomic framework.
Result(s): Our cross-sectional SLE cohort (N=51, age = 41.1+/-12.3, 100% female, 45% Hispanic, 24% black, 22% Asian, 51% white, SLEDAI = 4.4+/-4.2) was comprised of patients consecutively enrolled excluding those on aspirin or anticoagulants. Compared to the 18 controls, there were 2290 (p.adj < 0.05) differentially expressed genes. ( Figure 1 A, B) GSEA revealed positive enrichment for pathways related to interferon response, TNFa signaling, and coagulation in SLE. ( Figure 1C) WGCNA was used to create a modular transcriptomic framework. ( Figure 2A ) Modules enriched for platelet activity, immune response, and WNT signaling were significantly increased in SLE versus controls. Moreover, modules enriched for interferon response and WNT signaling paralleled increases in disease activity. ( Figure 2B) When analyzing patients with proteinuria, modules associated with oxidative phosphorylation and platelet activity were unexpectedly decreased. (Figure 2C) Analyzing the ratio of fold changes between SLE/Control vs SLE Proteinuria/SLE No Proteinuria, genes increased in SLE and those with proteinuria were enriched for immune effector processes, while genes increased in SLE but decreased in proteinuria were enriched for coagulation and cell adhesion. (Figure 2D) The module enriched for FCR activation was decreased in SLE and was affected by the FcgammaRIIa genotype. (Figure 3A) FcgammaRIIa R131 and H131 patients showed significantly different platelet transcriptomes. (Figure 3B) The combination of SLE with an FcgammaRIIa R131 variant leads to a significant increase in the platelet activity module not seen in controls. (Figure 3C)
Conclusion(s): These analyses reveal that SLE patients have a significantly different platelet transcriptome from controls, different phenotypic presentations of SLE patients associate with distinct platelet transcriptomic signatures, and FCGR2a variants may differentially influence the role of platelets in the contribution to SLE disease activity

Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury.

We have established experimental systems to assess the effects of early-life exposures to antibiotics on the intestinal microbiota and gene expression in the brain. This model system is highly relevant to human exposure and may be developed into a preclinical model of neurodevelopmental disorders in which the gut-brain axis is perturbed, leading to organizational effects that permanently alter the structure and function of the brain. Exposing newborn mice to low-dose penicillin led to substantial changes in intestinal microbiota population structure and composition. Transcriptomic alterations implicate pathways perturbed in neurodevelopmental and neuropsychiatric disorders. There also were substantial effects on frontal cortex and amygdala gene expression by bioinformatic interrogation, affecting multiple pathways underlying neurodevelopment. Informatic analyses established linkages between specific intestinal microbial populations and the early-life expression of particular affected genes. These studies provide translational models to explore intestinal microbiome roles in the normal and abnormal maturation of the vulnerable central nervous system.

Advances in mass-spectrometry have generated increasingly large-scale proteomics datasets containing tens of thousands of phosphorylation sites (phosphosites) that require prioritization. We develop a bioinformatics tool called HotPho and systematically discover 3D co-clustering of phosphosites and cancer mutations on protein structures. HotPho identifies 474 such hybrid clusters containing 1255 co-clustering phosphosites, including RET p.S904/Y928, the conserved HRAS/KRAS p.Y96, and IDH1 p.Y139/IDH2 p.Y179 that are adjacent to recurrent mutations on protein structures not found by linear proximity approaches. Hybrid clusters, enriched in histone and kinase domains, frequently include expression-associated mutations experimentally shown as activating and conferring genetic dependency. Approximately 300 co-clustering phosphosites are verified in patient samples of 5 cancer types or previously implicated in cancer, including CTNNB1 p.S29/Y30, EGFR p.S720, MAPK1 p.S142, and PTPN12 p.S275. In summary, systematic 3D clustering analysis highlights nearly 3,000 likely functional mutations and over 1000 cancer phosphosites for downstream investigation and evaluation of potential clinical relevance.

Glioblastoma (GBM) is the most aggressive nervous system cancer. Understanding its molecular pathogenesis is crucial to improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs provides insights to GBM biology. We identify key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation, as well as potential targets for EGFR-, TP53-, and RB1-altered tumors. Immune subtypes with distinct immune cell types are discovered using bulk omics methodologies, validated by snRNA-seq, and correlated with specific expression and histone acetylation patterns. Histone H2B acetylation in classical-like and immune-low GBM is driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identify specific lipid distributions across subtypes and distinct global metabolic changes in IDH-mutated tumors. This work highlights biological relationships that could contribute to stratification of GBM patients for more effective treatment.

BACKGROUND & AIMS/OBJECTIVE:Restorative proctocolectomy with ileal pouch-anal anastomosis is a surgical procedure in patients with ulcerative colitis refractory to medical therapies. Pouchitis, the most common complication, is inflammation of the pouch of unknown etiology. To define how the intestinal immune system is distinctly organized during pouchitis, we analyzed tissues from patients with and without pouchitis and from patients with ulcerative colitis using single-cell RNA sequencing (scRNA-seq). METHODS:We examined pouch lamina propria CD45+ hematopoietic cells from intestinal tissues of ulcerative colitis patients with (n=15) and without an ileal pouch-anal anastomosis (n=11). Further in silico meta-analysis was performed to generate transcriptional interaction networks and identify biomarkers for patients with inflamed pouches. RESULTS:In addition to tissue-specific signatures, we identified a population of IL1B/LYZ+ myeloid cells and FOXP3/BATF+ T cells that distinguish inflamed tissues which we further validated in other single cell RNA-seq datasets from IBD patients. Cell type specific transcriptional markers obtained from single-cell RNA-sequencing was used to infer representation from bulk RNA sequencing datasets, which further implicated myeloid cells expressing IL1B and S100A8/A9 calprotectin as interacting with stromal cells, and Bacteroidiales and Clostridiales bacterial taxa. We found that non-responsiveness to anti-integrin biologic therapies in ulcerative colitis patients was associated with the signature of IL1B+/LYZ+ myeloid cells in a subset of patients. CONCLUSIONS:Features of intestinal inflammation during pouchitis and ulcerative colitis are similar, which may have clinical implications for the management of pouchitis. scRNA-seq enables meta-analysis of multiple studies, which may facilitate the identification of biomarkers to personalize therapy for IBD patients.