Project description:Wiskott-Aldrich syndrome (WAS) is characterized by X-linked thrombocytopenia, eczema, immunodeficiency, recurrent infections and increased risk of autoimmunity and malignancies. WAS is caused by mutations in the WAS gene, which encodes the exclusively hematopoietic WAS protein (WASp) that is classically characterized as aÎ½ actin nucleator. However, disruption of F-actin polymerization by WAS mutations can not account for many aspects of WAS pathogenesis. Ignorance of other functions of WASP precludes in-depth understanding of the pathogenic effects of mutant WASP, and therefore hampers development of effective therapy. Here we generated induced pluripotent stem cells (iPSCs) from WAS patients (WAS-iPSC) bearing different mutations and corresponding isogenic iPSCs in which the pathogenic mutations had been corrected by targeted genome editing. Hematopoietic cells differentiated from WAS-iPSCs not only recapitulated known disease phenotypes, but also revealed novel defects of WASP deficient cells. WASP co-localized with nuclear pores, nucleoli, nuclear speckles and PML bodies by immunocytochemistry and/or serial block face scanning microscopy (SBF-SEM). MudPIT (multi-dimensional protein identification technology) analysis revealed that WASP physically interacted with nuclear body components, nuclear structural proteins, chromatin modifying complexes, and many RNA-binding proteins including major components of the spliceosome. Next-generation sequencing captured a dramatic global change of alternative splicing in WAS patient cells. WAS mutation impacted splicing of multiple genes frequently mutated in myelodysplastic syndrome and other cancers. RNA sequencing showed that WAS-iPSC derived immune cells misregulated many cell cycle regulators, tumor suppressors, immune function genes and splicing factors, and activated gene networks that drive cancer development and inflammatory diseases. Together these data uncovered previously unappreciated functions of the WASP and provided a mechanistic understanding of the pathogenesis of malignancy and autoimmunity in the most severe form of WAS. These new knowledge could help develop targeted therapy for WAS in the future. Human WAS-iPSCs (p.Phe35*) and gene corrected WAS-iPSCs (cWAS-iPSC) were differentiated into macrophages. WAS patient derived B-lymphocyte line ID00003 (p.Glu133Lys) and a wile-type B-lymphocyte line GM11518 were cultured using standard protocol. Total RNAs were extracted and been analyzed by RASL-seq.

Project description:Phagocytosis requires the activation of a plethora of mechanisms that include the activation of the actin cytoskeleton guided by the Arp2/3 complex. These are promoted by activators such as the Wiskott Aldrich Syndrome Protein (WASP) family members. In order to further understand the molecular mechanisms involved in the early events leading the phagocytosis of the pathogenic Mycobacterium tuberculosis, we set out to examine potential roles of miRNAs in phagocytosis using genome-wide expression profiling to identify miRNAs differentially regulated following mycobacterial infection. One of the miRNAs activated upon infection of mouse macrophages with the non-pathogenic Mycobacterium smegmatis, the widely conserved miR-142-3p, was predicted and confirmed to target the Neural-WASP (N-WASP). Upregulating of miR-142-3p in mouse macrophages inversely correlated with levels of N-WASP, upon infection with live pathogenic and non-pathogenic mycobacteria, suggesting an active role of Mycobacterium tuberculosis on the regulation of phagocytosis, at the post-transcriptional level, in host cells. The reduction of N-WASP correlated with a reduced internalization of bacteria per macrophage, independently of the phagocytosis index. Furthermore, the downregulation of WASP levels accompanied those of N-WASP, at early but not at late time points, suggesting a closely regulatory mechanism among both family members, dependent on the time frame of the phagocytosis. Additionally, upregulating of miR-142-3p promoted the change in the protein levels of another predicted and confirmed target, the Cofilin2 protein, in a phagocytosis-independent fashion. Downregulation experiments promoted aberrant morphologic phenotypes in macrophages, similar to observed by others in PBMCs of humans with Wiskott Aldrich Syndrome, suggesting the strong involvement of miR-142-3p on the regulation of the actin machinery in macrophages. Altogether these results show for the first time that miRNAs are involved in the regulation of actin-mediated phagocytosis of pathogenic bacteria and that these are direct targets of Mycobacterium tuberculosis.

Project description:Phagocytosis requires the activation of a plethora of mechanisms that include the activation of the actin cytoskeleton guided by the Arp2/3 complex. These are promoted by activators such as the Wiskott Aldrich Syndrome Protein (WASP) family members. In order to further understand the molecular mechanisms involved in the early events leading the phagocytosis of the pathogenic Mycobacterium tuberculosis, we set out to examine potential roles of miRNAs in phagocytosis using genome-wide expression profiling to identify miRNAs differentially regulated following mycobacterial infection. One of the miRNAs activated upon infection of mouse macrophages with the non-pathogenic Mycobacterium smegmatis, the widely conserved miR-142-3p, was predicted and confirmed to target the Neural-WASP (N-WASP). Upregulating of miR-142-3p in mouse macrophages inversely correlated with levels of N-WASP, upon infection with live pathogenic and non-pathogenic mycobacteria, suggesting an active role of Mycobacterium tuberculosis on the regulation of phagocytosis, at the post-transcriptional level, in host cells. The reduction of N-WASP correlated with a reduced internalization of bacteria per macrophage, independently of the phagocytosis index. Furthermore, the downregulation of WASP levels accompanied those of N-WASP, at early but not at late time points, suggesting a closely regulatory mechanism among both family members, dependent on the time frame of the phagocytosis. Additionally, upregulating of miR-142-3p promoted the change in the protein levels of another predicted and confirmed target, the Cofilin2 protein, in a phagocytosis-independent fashion. Downregulation experiments promoted aberrant morphologic phenotypes in macrophages, similar to observed by others in PBMCs of humans with Wiskott Aldrich Syndrome, suggesting the strong involvement of miR-142-3p on the regulation of the actin machinery in macrophages. Altogether these results show for the first time that miRNAs are involved in the regulation of actin-mediated phagocytosis of pathogenic bacteria and that these are direct targets of Mycobacterium tuberculosis. Expression analysis of mouse macrophage cell line J774A.1 in response to infection with Mycobacterium smegmatis mc2155 EGFP. Three biological replicates each for uninfected and infected samples.

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Proteome quantification in WASP wild-type and deficient macrophages using DIA-MS

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