Project description:The complex system by which the skin regulates immune responses to the external environment is unclear. Here, we investigated cell-cell interactions underlying cutaneous defense against S. aureus. Single-cell transcriptomics (scRNA-Seq) and unbiased network analysis revealed unexpected, dominant IL-17-mediated dermal reticular fibroblast-to-neutrophil communication. Multi-faceted in vitro omics studies demonstrated that IL-17 synergized with several factors including TNF⍺ to induce fibroblast NFKBIZ and chemokine secretion. Cultured fibroblasts drove robust neutrophil recruitment through NFKBIZ-dependent CXCR2 and CXCR4 ligands. Mice lacking IL-17R in fibroblasts (PdgfraΔIl17ra) were generated to determine the significance of fibroblast-neutrophil communication. PdgfraΔIl17ra mice exhibited drastically reduced skin neutrophilia in multiple disease models and reduced defense against S. aureus. These findings were translated to humans by comprehensive analysis of biopsies from psoriasis patients on and off anti-IL-17 treatment. Thus, dermal fibroblasts are critical for skin type 17 inflammation and represent a novel target for treatment of infection and inflammatory disease.
Project description:The complex system by which the skin regulates immune responses to the external environment is unclear. Here, we investigated cell-cell interactions underlying cutaneous defense against S. aureus. Single-cell transcriptomics (scRNA-Seq) and unbiased network analysis revealed unexpected, dominant IL-17-mediated dermal reticular fibroblast-to-neutrophil communication. Multi-faceted in vitro omics studies demonstrated that IL-17 synergized with several factors including TNF⍺ to induce fibroblast NFKBIZ and chemokine secretion. Cultured fibroblasts drove robust neutrophil recruitment through NFKBIZ-dependent CXCR2 and CXCR4 ligands. Mice lacking IL-17R in fibroblasts (PdgfraΔIl17ra) were generated to determine the significance of fibroblast-neutrophil communication. PdgfraΔIl17ra mice exhibited drastically reduced skin neutrophilia in multiple disease models and reduced defense against S. aureus. These findings were translated to humans by comprehensive analysis of biopsies from psoriasis patients on and off anti-IL-17 treatment. Thus, dermal fibroblasts are critical for skin type 17 inflammation and represent a novel target for treatment of infection and inflammatory disease.
Project description:The hair follicle (HF) is a complex miniorgan that serves as an ideal model system to study stem cell (SC) interactions with the niche during growth and regeneration. Dermal papilla (DP) cells are required for activating SCs during the adult hair cycle, but the signal exchange between niche and SC precursors/transit amplifying progenitor cells (TACs) that regulates HF morphogenetic growth is largely unknown. Here we use six transgenic reporters to isolate 14 major skin and HF cell populations. With next-generation RNA sequencing we characterize their transcriptomes and define unique molecular signatures. SC precursors, TACs and the DP niche express a plethora of known and novel ligands and receptors. Signaling interaction network analysis reveals a birds-eye view of pathways implicated in epithelial-mesenchymal interactions. Using a systematic tissue-wide approach this work provides a comprehensive platform, linked to an interactive online database, to identify and further explore the SC/TAC/niche crosstalk regulating HF growth.
Project description:The transition to flowering in plants is controlled by a regulatory network that responds to both developmental and environmental signals. The MADS-box genes FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) are major flowering repressors that enhance responses to environmental cues such as winter temperatures, high ambient temperatures and photoperiod. FLC and SVP physically interact in vivo and mutation of each gene causes early flowering while the double mutant is more extreme. The molecular mechanisms underlying these genetic interactions are mostly unknown. We addressed the regulatory input of these two key transcription factors (TFs) both individually and as a complex at the genome-wide level through ChIP-seq and microarray expression analysis in single and double mutants. Analysis of each TF demonstrated that the complex acts predominantly via functional redundancy in the repression of flowering. SVP and FLC bind to the same regions of the flowering genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT) but do not require the presence of the other to bind. However, genome-wide identification of SVP and FLC occupancy events revealed that their binding scenarios are quantitatively and qualitatively affected by the presence of the cognate partner. A subgroup of genes whose regulation by these TFs depends exclusively on combinatorial binding of both proteins was identified, demonstrating a qualitatively essential role of the SVP-FLC complex. Some of these genes are involved in the control of flowering through direct and indirect regulation of Gibberellin-related processes. Cis-regulatory elements enriched only at such complex-bound sites were identified. Thus the regulatory output mediated by SVP and FLC reveals substantial flexibility, leading to dependent and independent DNA binding that enables additive, cooperative and repressive modes of co-regulation. In total 48 samples; 24 leaf samples corresponding to two different growing conditions, 24 apex samples corresponding to two different growing conditions.
Project description:Gene expression analysis of pancreatic cancer associated fibroblasts and control fibroblasts To identify signaling pathways important in tumor-stromal cell interactions, we performed gene expression profiling on pancreatic cancer associated fibroblast cultures and control fibroblast cultures using Affymetrix Exon arrays.
Project description:Understanding the molecular interactions of host cells with Ebola virus (EBOV) is integral for further therapeutic development of antivirals. We performed a proximity-dependent protein interaction screen for six of seven EBOV proteins to characterize the EBOV-host interactome. Using network analysis, putative EBOV interacting proteins were mapped to a human protein interactome. We overlayed viral protein interactions onto the host network. Within this map, several known EBOV-host protein interactions were identified, as well as several cell processes, such as RNA processing, previously implicated in EBOV replication. Furthermore, this interactome revealed novel interactions between EBOV proteins and host proteins arranged in functional complexes. As proof of this concept, we characterized the interaction between EBOV VP35 and the mRNA decapping complex, which regulates mRNA turnover in host cells. Our protein-protein interaction data demonstrate that VP35 engages multiple components of this complex by binding binds the scaffold protein EDC4. Inhibiting expression of mRNA decapping complex components EDC4, EDC3, or DCP2 inhibited viral replication by reducing early viral RNA synthesis. Our approach examining EBOV-host protein interactions in conjunction with network analysis reveals how EBOV interacts with entire cellular machines as opposed to singular cell proteins to promote its replication.
Project description:Gene expression analysis of pancreatic cancer associated fibroblasts and control fibroblasts To identify signaling pathways important in tumor-stromal cell interactions, we performed gene expression profiling on pancreatic cancer associated fibroblast cultures and control fibroblast cultures using Affymetrix Exon arrays. We analyzed 3 control fibroblast cultures and 9 pancreatic cancer associated fibroblast cultures using the Affymetrix Human Exon 1.0 ST platform. Gene expression levels were compared using Partek (version 6.3beta).
Project description:Antibiotic biosynthesis in Streptomyces species is controlled by a complex genetic and biochemical network of global and pathway specific regulators. Details of their precise interactions in mediating temporal and spatial expression of secondary metabolite genes remain poorly defined. In this study, we employed whole-genome microarrays to investigate the temporal transcriptome profiles of S. coelicolor A3(2) deltaSCO2517::kan mutant strain (LY2517) and compare it to the wild-type M145 strain. SCO2517 is a putative two-component system response regulator. The time-course study involved analysis of 6 samples (15h, 22h, 24h, 26h, 35h, 43h). All samples were analyzed on duplicate arrays (2 technical replicates). Genomic DNA of S. coelicolor wild type (M145) was used as a reference for all the arrays. cDNA was labeled with Alexa647 and genomic DNA was labeled with Cy3.