The federal Project Grant award R01HL178823, funded by the National Heart, Lung, and Blood Institute (NHLBI) under the Cardiovascular Diseases Research program (CFDA 93.837), is focused on exploring therapeutic strategies for treating and preventing neutropenia-related bacterial pneumonia. The $676,748 award aims to increase the lifespan of neutrophils in the lungs of neutropenic patients by targeting the cell death executor Gasdermin D (GSDMD). The research seeks to uncover the underlying...
This $959,457.45 Project Grant award from the National Heart, Lung, and Blood Institute (NHLBI), under the Cardiovascular Diseases Research program (CFDA 93.837), supports research to investigate the role of the transcription factor THPOK in regulating myelopoiesis, the development of myeloid cells. The research aims to: Define how growth factor and cytokine receptor signaling regulates THPOK expression in myeloid progenitors. Elucidate the mechanism by which THPOK regulates gene expression...
This $423,000 Project Grant was awarded by the National Heart Lung and Blood Institute (NHLBI) under the Cardiovascular Diseases Research federal grant program (CFDA 93.837). The grant supports research to investigate the direct regulation of cell cycle genes by cell identity factors during myeloid differentiation. Specifically, the project aims to determine how transcription factors PU.1 and C/EBP regulate the expression of cyclin-dependent kinase inhibitors and cyclin D2 to induce cell cycle...
The federal Project Grant award of $445,000.00 from the National Heart, Lung, and Blood Institute (NHLBI), under the Cardiovascular Diseases Research Program (CFDA 93.837), will support research to understand the mechanisms by which mutations in the CLPB gene lead to impaired neutrophil development and severe congenital neutropenia (SCN). The research aims to define how mitochondrial dysfunction caused by CLPB mutations leads to neutrophil precursor cell death, and how somatic RUNX1 mutations...
The National Institutes of Health Office of the Director awarded a $2,925,000 Project Grant to The Trustees of the University of Pennsylvania under the Trans-NIH Research Support program (CFDA 93.310) on September 1, 2022 for a three-year period ending August 31, 2025. The grant will fund research to develop an in-depth understanding of transcriptional and epigenetic reprogramming of neutrophils in the tumor microenvironment. The grantee will leverage single-cell profiling, fate mapping, and...
The National Heart, Lung, and Blood Institute (NHLBI) awarded a 3-year, $445,000 Project Grant under the Cardiovascular Diseases Research program (CFDA 93.837) to The Children's Hospital of Philadelphia (CHOP) Research Institute. The grant is for a research project titled "NET STABILIZATION: MECHANISTIC AND THERAPEUTIC STUDIES STUDYING THE ROLE OF NETS IN THROMBOINFLAMMATORY DISEASE." The project aims to characterize the role of neutrophil extracellular traps (NETs) in immune...
This federal Project Grant award from the National Heart, Lung, and Blood Institute (NHLBI), under the Cardiovascular Diseases Research program (CFDA 93.837), provides $179,280 to The Trustees of the University of Pennsylvania to investigate how early-life nutritional stress impacts neutrophil development and function, and how this may contribute to the severity of sickle cell disease. The research aims to evaluate the correlation between diet, disease severity, and neutrophil activity in sickle...
This federal Project Grant award for $657,368.00, issued by the National Heart Lung and Blood Institute (NHLBI) under the Cardiovascular Diseases Research program (CFDA 93.837), supports research to investigate the mechanisms controlling microRNA (miRNA) processing and degradation pathways during hematopoietic development. The primary awardee, The Washington University, will utilize human pluripotent stem cell lines with mutations in miRNA deadenylases and exosome components to identify novel...
This $693,942 National Institutes of Health National Heart Lung and Blood Institute Project Grant funds research at Northwestern University aimed at elucidating epigenetic modifiers of regulatory T cell function following viral pneumonia. The goal is to establish causal links between DNA methyltransferase activity, the DNMT adapter UHRF1, and DNA hypermethylation signatures in regulatory T cells during aging and their impaired reparative function after severe viral pneumonia. Through three...
This $444,952 federal Project Grant award, titled "Exploring the Impact of Base Deaminase Deregulation on Precancer Evolution," was provided by the National Cancer Institute (CFDA 93.396 Cancer Biology Research) to the University of California, San Diego (UC San Diego). The goal of this 5-year research project is to investigate how deregulation of the APOBEC3C and ADAR1 base deaminase enzymes drives the transformation of pre-leukemic stem cells (pre-LSCs) into more aggressive...
IDENTIFICATION OF NOVEL REGULATORS FOR NEUTROPHIL DEVELOPMENT AND LIFESPAN - ABSTRACT: MY LABORATORY IS INTERESTED IN STUDYING SIGNALING MECHANISMS FOR THE REGULATION OF NEUTROPHIL BIOLOGY. NEUTROPHILS ARE A TYPE OF POLYMORPHONUCLEAR LEUKOCYTES AND ARE WELL RECOGNIZED AS ONE OF THE MAJOR PLAYERS DURING INFLAMMATION. ALTHOUGH NEUTROPHILS ARE REQUIRED FOR NORMAL IMMUNE RESPONSES, THEY ARE ALSO RESPONSIBLE FOR MANY INFLAMMATION-RELATED DISEASES AS THE EFFECTOR MOLECULES RELEASED BY NEUTROPHILS ARE TOXIC TO HOST TISSUES. MATURE NEUTROPHILS ARE KNOWN FOR THEIR SHORT LIFESPAN (HALF-LIFE IS GENERALLY BELIEVED LESS THAN A DAY), AND THEIR LIFESPAN MAY BE EXPANDED IN INFLAMMATORY AND DISEASE CONDITIONS. THUS, THE CONTROL OF NEUTROPHIL DEVELOPMENT AND LIFESPAN CAN BE A KEY STEP IN REGULATING NEUTROPHIL-RELATED FUNCTIONS IN NORMAL PHYSIOLOGY AS WELL AS DISEASE CONDITIONS. HOWEVER, NOT MUCH IS KNOWN ABOUT THE MECHANISMS UNDERLYING NEUTROPHIL DEVELOPMENT AND LIFESPAN REGULATION. WHILE TRANSCRIPTION FACTORS LIKE CEBPE ARE KNOWN TO PLAY A ROLE IN NEUTROPHIL DEVELOPMENT, INVESTIGATIONS INTO THESE MECHANISMS HAVE BEEN LIMITED TO INDIVIDUAL STUDIES. THEREFORE, A COMPREHENSIVE GENETIC SCREEN WOULD PROVIDE COMPREHENSIVE KNOWLEDGE OF NEUTROPHIL DEVELOPMENT. IN ADDITION, ALTHOUGH VARIOUS DEATH MECHANISMS, PARTICULARLY APOPTOSIS, PLAY ROLES IN NEUTROPHIL DEATH, THEIR BLOCKADES ONLY HAD MODERATE EFFECTS, SUGGESTING UNKNOWN MECHANISMS MAY EXIST. THUS, WE HYPOTHESIZE THAT THERE MIGHT BE NOVEL MECHANISMS FOR THE REGULATION OF NEUTROPHIL LIFESPAN. PRIMARY NEUTROPHILS HAVE A VERY SHORT LIFESPAN AND ARE UNABLE TO EXPAND PERPETUALLY IN VITRO. TOGETHER WITH THEIR BEING REFRACTORY TO TRANSFECTION OR VIRAL TRANSDUCTION, NEUTROPHILS HAVE NEVER BEEN USED IN ANY LARGE-SCALE GENETIC SCREEN IN MICE. THE HOXB8-ER FUSION PROTEIN HAS BEEN USED TO IMMORTALIZE MOUSE HEMATOPOIETIC PROGENITOR CELLS (HOXB8-HPCS), WHICH CAN DIFFERENTIATE INTO NEUTROPHILS BOTH IN VITRO AND IN VIVO. THE HOXB8-HPC-DERIVED NEUTROPHILS WERE FUNCTIONALLY SIMILAR TO FRESHLY ISOLATED MOUSE NEUTROPHILS. BECAUSE HOXB8-HPCS CAN BE UNLIMITED EXPANDED AND TRANSDUCED WITH LENTIVIRUS, THEY ARE AMENABLE TO GENETIC SCREENS. IN OUR PRELIMINARY STUDIES, WE HAVE ESTABLISHED HOXB8-HPCS FROM A CAS9 KNOCK-IN MOUSE LINE. THESE CELLS CAN BE TRANSDUCED WITH LENTIVIRUS WITH SUFFICIENT EFFICIENCY TO MEET OUR SCREENING GOALS. WE ALSO DID A PILOT IN VIVO SCREEN USING ONE OF THE SUB-LIBRARIES, WHICH DEMONSTRATED THE FEASIBILITY OF OUR SCREEN STRATEGY AND METHOD. THEREFORE, IN THIS EXPLORATORY R21 PROPOSAL, WE PROPOSE TO PERFORM THE FIRST CRISPR-CAS9-BASED SCREENS FOR KEY REGULATORS THAT CONTROL NEUTROPHIL DIFFERENTIATION FROM HOXB8-HPCS AND LIFESPAN OF DIFFERENTIATED NEUTROPHILS IN MICE. OUR PROPOSED WORK WILL NOT ONLY ESTABLISH A VALUABLE RESEARCH TOOL BUT ALSO YIELD INSIGHTS INTO NEUTROPHIL BIOLOGY THAT CANNOT BE PREDICTED FROM EXISTING KNOWLEDGE. IMPORTANTLY, THE TOOL WE ARE DEVELOPING AND VALIDATING HERE CAN BE READILY ADAPTED FOR SCREENS WITH MANY OTHER POSSIBLE READOUTS.