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Updated: Jan 16, 2026
Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Published on: August 2, 2021
PSTPIP1 and pyrin, two key regulators of macrophage differentiation
Philipp Berger1, Lisa Wilming2, Ricarda Jürgens2
1Institute of Immunology, University of Muenster, Muenster, Germany; Clinic for Paediatric and Adolescent Medicine, University Hospital RWTH Aachen, Aachen, Germany.
Background:
Monocytes develop from hematopoietic stem cells; migrate into the tissue, where they undergo a stimulation-dependent and tissue specific differentiation into macrophages imprinting specific inflammatory functions. The development of inflammatory functions during differentiation of progenitor cells into macrophages remained incompletely understood.
Objective:
We intended to identify regulatory factors driving monocyte/macrophage differentiation.
Methods:
A Genome-wide CRISPR/Cas9 knockout screen (GeCKO) in ER-HoxB8 macrophages was used to identify key drivers of macrophage differentiation which were verified in independent knock-out and knock-in cells. Immunophenotyping was studied by FACS, morphology and migration by fluorescence microscopy, the inflammatory response by ELISA. Transcriptomic data were obtained by next generation mRNA sequencing and validated by quantitative polymerase chain reaction and immunoblotting.
Results:
Genome-wide CRISPR/Cas9 knockout screen identified the cytosolic cytoskeleton-associated adaptor molecule PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1) as a regulatory factor of macrophage differentiation. Interestingly, mutations in PSTPIP1 cause autoinflammatory disorders (PAPA syndrome). Deletion of PSTPIP1 resulted in hampered differentiation, decreased inflammatory response, changed morphology, altered cell adhesion and migration properties. PSTPIP1 is a regulator of Pyrin inflammasome activity which drives autoinflammation in familial Mediterranean fever (FMF). Deletion of Pyrin also resulted in a strong alteration of cellular dynamics in macrophages.
Conclusion:
PSTPIP1 and Pyrin are crucial factors in macrophage differentiation. Their deletion or mutation resulted in a hampered differentiation of macrophages resulting in strong morphological alterations and impacting phagocyte key functions as adhesion and migration. Impaired differentiation of macrophages may represent a significant factor in the pathophysiology of autoinflammatory diseases like FMF and PAPA.
Insights
Proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) and Pyrin are key regulators of macrophage differentiation. Impaired PSTPIP1 or Pyrin function hampers differentiation, affecting immune cell function and potentially contributing to autoinflammatory diseases.
Area of Science:
- Immunology
- Cell Biology
- Genetics
Background:
- Monocytes differentiate into tissue-specific macrophages, acquiring inflammatory functions.
- The precise mechanisms governing macrophage differentiation remain incompletely understood.
Purpose of the Study:
- To identify regulatory factors controlling monocyte to macrophage differentiation.
- To elucidate the role of identified factors in macrophage function and inflammatory responses.
Main Methods:
- Genome-wide CRISPR/Cas9 knockout screening (GeCKO) in ER-HoxB8 macrophages.
- Validation using knock-out/knock-in cells, immunophenotyping (FACS, microscopy), inflammatory response assays (ELISA), and transcriptomics (mRNA sequencing, qPCR).
Main Results:
- PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1) was identified as a critical regulator of macrophage differentiation.
- PSTPIP1 deletion impaired differentiation, reduced inflammatory response, altered cell morphology, adhesion, and migration.
- PSTPIP1 regulates Pyrin inflammasome activity, a key driver of autoinflammation in diseases like Familial Mediterranean Fever (FMF).
- Pyrin deletion also significantly altered macrophage cellular dynamics.
Conclusions:
- PSTPIP1 and Pyrin are essential for proper macrophage differentiation and function.
- Defects in PSTPIP1 or Pyrin lead to hampered macrophage differentiation, impacting phagocyte functions.
- Impaired macrophage differentiation may play a significant role in the pathophysiology of autoinflammatory disorders such as FMF and PAPA syndrome.
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