Effects of STAT3 gain of function on myeloid cells in human peripheral blood mononuclear cells

Abstract

Background: Monogenic germline mutations in signal transducer and activator of transcription 3 (STAT3) can cause gain of function (GOF) characterised by hyperactivity of the transcription factor and an increase in transcription of STAT3 target genes. Mutations can be hereditary or sporadic and occur in the various functional domains of the STAT3 gene product. The disease presents early in life with IPEX‐like, ALPS‐like, and STAT5 deficiency‐like symptoms, multisystem autoimmunity and immune deficiency. The phenotype is best described as STAT3 GOF induced immune dysregulation and has severe clinical implications. To date there remains a paucity of research into this condition and an unmet clinical need meaning this research could aid understanding of this very complex condition.Objective: To assess how STAT3 GOF mutations affect gene expression, cell populations, cytokine production and to identify the mechanism of hyperactivity. Approaches: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood obtained from STAT3 GOF patients and healthy control and profiled using single cell RNA sequencing. In addition, the myeloid compartment of these donors were phenotyped using Trucount and sequenced using mini‐bulk RNA sequencing to confirm observations from single cell work. Furthermore, inducible pluripotent stem cell (IPSC) derived macrophages were used to validate observations made during RNA sequencing experiments. Finally, primary human dermal fibroblasts from STAT3 GOF patients and healthy controls were used to identify the mechanism of hyperactivity of STAT3 GOF mutants. Key findings: Single cell RNA sequencing of PBMCs identified 17 populations in total including all major myeloid cells, DCs and T‐cells as well as identifying increased expression of glycolytic genes in STAT3 GOF samples. Phenotyping of the myeloid compartment identified all monocyte and dendritic cell (DC) populations but with some reductions in some DC populations in STAT3 GOF. Mini‐bulk RNA sequencing of the myeloid compartment confirmed an increase in the expression of genes related to glycolysis. Validation studies demonstrated STAT3 GOF IPSC’s were able to generate mature functional macrophages that displayed altered glycolytic characteristics as well as altered response to lipopolysaccharide (LPS) challenge compared to controls. Mechanistic investigations revealed no altered protein stability or phosphorylation kinetics with STAT3 GOF but a retention of STAT3 in the nucleus was observed. Preliminary data suggests that STAT3 GOF mutations may not increase DNA binding capacity but may alter acetylation kinetics. Conclusion: STAT3 GOF mutations do not appear to affect development or maturation of PBMC populations but may alter some DC population numbers possibly through ii sequestration. Moreover, metabolic investigations suggest that STAT3 GOF may perturb glycolysis and LPS response in IPSC derived macrophages, but the mechanism remains unknown. Finally, preliminary mechanistic data reveals a possible mode of action for STAT3 GOF via altered acetylation kinetics which may increase transcriptional activity of STAT3 several fold.