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Janus Kinase Inhibitor Brepocitinib Rescues Myelin Phagocytosis Under Inflammatory Conditions: In Vitro Evidence from Microglia and Macrophage Cell Lines

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Abstract

Central nervous system (CNS) injuries induce cell death and consequently the release of myelin and other cellular debris. Microglia as well as hematogenous macrophages actively collaborate to phagocyte them and undergo their degradation. However, myelin accumulation persists in the lesion site long after the injury with detrimental effects on axonal regeneration. This might be due to the presence of inhibitors of phagocytosis in the injury site. As we recently published that some proinflammatory stimuli, like interferon-γ (IFNγ) and lipopolysaccharide (LPS), inhibit myelin phagocytosis in macrophages, we have now studied the signaling pathways involved. A phagocytosis assay in Raw264.7 macrophages and N13 microglia cell lines with labeled myelin was developed with the pHrodo reagent that emits fluorescence in acidic cellular compartments (e.g.lysosomes). Pharmacological inhibition of Janus kinases (Jak) with Brepocitinib restored myelin phagocytosis and rescued the expression of genes related to phagocytosis, like triggering receptor expressed on myeloid cells 2 (TREM2), induced by IFNγ or LPS. In addition, while pharmacological inhibition of the signal transducer and activator of transcription 3 (STAT3) rescued myelin phagocytosis and the expression of phagocytosis related genes in the presence of LPS, it did not have any effect on IFNγ-treated cells. Our results show that Jak pathways participate in the inhibition of myelin phagocytosis by IFNγ and LPS. They also indicate that the resolution of inflammation is important for the clearance of cellular debris by macrophages and subsequent regenerative processes.

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Supporting data are included in the Supplementary Information. All other data are available from the corresponding author upon reasonable request.

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Acknowledgements

We appreciate the expert assistance provided by the HNP core facility of Microscopy.

Funding

This work was funded by Grants SAF2017-89366-R (MINECO/AEI/FEDER/EU) and PID2021-128446NB-I00 (MCIN/AEI/https://doi.org/10.13039/501100011033 and by “ERDF A way of making Europe”, by the “European Union”). We appreciate the expert assistance provided by the HNP core facility of Microscopy.

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Authors and Affiliations

  1. Laboratorio de Regeneración Neuronal, Hospital Nacional de Parapléjicos, SESCAM, Finca La Peraleda S/N, 45071, Toledo, Spain

    Lorenzo Romero-Ramírez, Concepción García-Rama & Jörg Mey

  2. School of Mental Health and Neuroscience, Maastricht University, Maastricht, Netherlands

    Jörg Mey

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  1. Lorenzo Romero-Ramírez
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Contributions

Lorenzo Romero-Ramírez: designed and supervised the study; performed the experiments, analyzed the data and wrote the manuscript. Concepción García-Rama: performed the experiments. Jörg Mey: supervised the study, drew Fig. 8 and reviewed the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Lorenzo Romero-Ramírez.

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Romero-Ramírez, L., García-Rama, C. & Mey, J. Janus Kinase Inhibitor Brepocitinib Rescues Myelin Phagocytosis Under Inflammatory Conditions: In Vitro Evidence from Microglia and Macrophage Cell Lines. Mol Neurobiol (2024). https://doi.org/10.1007/s12035-024-03963-6

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