Dataset Information

Data in support of Absence of miRNA-146a differentially alters microglia function and proteome

ABSTRACT: MiR-146a is an important regulator of innate inflammatory responses and is also implicated in cell death and survival. Here, we identified microglia as the main cellular source of miR-146a among mouse CNS resident cells. We further characterized the phenotype of miR-146a KO microglia cells during in vivo demyelination induced by cuprizone (CPZ) and found reduced number of CD11c+ microglia in the KO compared to WT mice. Microglia were also isolated from the brain, and the proteome was analyzed by liquid chromatography mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Microglial Cell

SUBMITTER: Allan Stensballe

LAB HEAD: Allan Stensballe

PROVIDER: PXD015939 | Pride | 2020-06-29

REPOSITORIES: Pride

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Absence of miRNA-146a Differentially Alters Microglia Function and Proteome.

Martin Nellie A NA Hyrlov Kirsten H KH Elkjaer Maria L ML Thygesen Eva K EK Wlodarczyk Agnieszka A Elbaek Kirstine J KJ Aboo Christopher C Okarmus Justyna J Benedikz Eirikur E Reynolds Richard R Hegedus Zoltan Z Stensballe Allan A Svenningsen Åsa Fex ÅF Owens Trevor T Illes Zsolt Z

Frontiers in immunology 20200605

<b>Background:</b> MiR-146a is an important regulator of innate inflammatory responses and is also implicated in cell death and survival. <b>Methods:</b> By sorting CNS resident cells, microglia were the main cellular source of miR-146a. Therefore, we investigated microglia function and phenotype in miR-146a knock-out (KO) mice, analyzed the proteome of KO and wild-type (WT) microglia by LC-MS/MS, and examined miR-146a expression in different brain lesions of patients with multiple sclerosis (MS ...[more]

PMID: 32582192

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Project description:Microglia are the resident mononuclear phagocytes of the CNS parenchyma and represent an initial line of defense against invading microorganisms. Microglia utilize Toll-like receptors (TLRs) for pathogen recognition and TLR2 specifically senses conserved motifs of gram-positive bacteria including lipoproteins, lipoteichoic acids, and peptidoglycan (PGN) leading to cytokine/chemokine production. Interestingly, primary microglia derived from TLR2 knockout (KO) mice over-expressed numerous IL-12 family members, including IL-12p40, IL-12p70, and IL-27 in response to intact S. aureus, but not the less structurally complex TLR2 ligands Pam3CSK4 or PGN. The ability of intact bacteria to augment IL-12 family member expression was specific for gram-positive organisms, since numerous gram-negative strains were unable to elicit exaggerated responses in TLR2 KO microglia. Inhibition of SYK or IRAK4 signaling did not impact heightened IL-12 family member production in S. aureus-treated TLR2 KO microglia, whereas PI3K, MAPK, and JNK inhibitors were all capable of restoring exaggerated cytokine expression to wild type levels. Additionally, elevated IL-12 production in TLR2 KO microglia was ablated by a TLR9 antagonist, suggesting that TLR9 drives IL-12 family member production following exposure to intact bacteria that remains unchecked in the absence of TLR2 signaling. Collectively, these findings indicate crosstalk between TLR2 and TLR9 pathways to regulate IL-12 family member production by microglia. The summation of TLR signals must be tightly controlled to ensure the timely cessation and/or fine tuning of cytokine signaling to avoid nonspecific bystander damage due to sustained IL-12 release. TLR2 KO mice were backcrossed with C57BL/6 animals for a minimum of eight generations prior to use in these studies. Age- and sex-matched C57BL/6 mice were used as wild type (WT) controls. Primary mixed glial cultures were prepared from the cerebral corticies of neonatal mice (2-4 days of age) and microglia were harvested using a differential shaking technique with a purity of >98%. A USA300 community-acquired methicillin-resistant S. aureus (CA-MRSA) clinical isolate recovered from a patient with a fatal brain abscess was used to stimulate the microglia isolates. Bacterial strains were heat-inactivated and used to stimulate microglia at 107 colony forming units (cfu)/well for 6 and 12 hours time points. Three replicates of each mouse type (WT, TLR2 KO) at both time points 6 and 12 hours were used for the microarray experiments. Data was only usable for 2 replicates of the KO-12 hr group.

Project description:MiR-142 is dynamically expressed and plays a regulatory role in hematopoiesis. Based on the simple observation that miR-142 levels are significantly lower in CD34+CD38- cells from blast crisis (BC) chronic myeloid leukemia (CML). CML patients compared with chronic phase (CP) CML patients (p=0.002), we hypothesized that miR-142 deficit plays a role in BC transformation. To test this hypothesis, we generated a miR-142 KO BCR-ABL (i.e., miR-142−/−BCR-ABL) mouse by crossing a miR-142−/− mouse with a miR-142+/+BCR-ABL mouse. While the miR-142+/+BCR-ABL mice developed and died of CP CML, the miR-142−/−BCR-ABL mice developed a BC-like phenotype in the absence of any other acquired gene mutations and died significantly sooner than miR-142+/+BCR-ABL CP controls (p=0.001). Leukemic stem cell (LSC)-enriched Lineage-Sca-1+c-Kit+ cells (LSKs) from diseased miR-142−/−BCR-ABL mice transplanted into congenic recipients, recapitulated the BC features thereby suggesting stable transformation of CP-LSCs into BC-LSCs in the miR-142 KO CML mouse. Single cell (sc) RNA-seq profiling showed that miR-142 deficit changed the cellular landscape of the miR-142−/−BCR-ABL LSKs compared with miR-142+/+BCR-ABL LSKs with expansion of myeloid-primed and loss of lymphoid-primed factions. Bulk RNA-seq analyses along with unbiased metabolomic profiling and functional metabolic assays demonstrated enhanced fatty acid β-oxidation (FAO) and oxidative phosphorylation (OxPhos) in miR-142−/−BCR-ABL LSKs vs miR-142+/+BCR-ABL LSKs. MiR-142 deficit enhanced FAO in miR-142−/−BCR-ABL LSKs by increasing the expression of CPT1A and CPT1B, that controls the cytosol-to-mitochondrial acyl-carnitine transport, a critical step in FAO. MiR-142 deficit also enhanced OxPhos in miR-142−/−BCR-ABL LSKs by increasing mitochondrial fusion and activity. As the homeostasis and activity of LSCs depend on higher levels of these oxidative metabolism processes, we then postulate that miR-142 deficit is a potentially druggable target for BC-LSCs. To this end, we developed a novel CpG-miR-142 mimic oligonucleotide (ODN; i.e., CpG-M-miR-142) that corrected the miR-142 deficit and alone or in combination with a tyrosine kinase inhibitor (TKI) significantly reduced LSC burden and prolonged survival of miR-142−/−BCR-ABL mice. The results from murine models were validated in BC CD34+CD38- primary blasts and patient-derived xenografts (PDXs). In conclusion, an acquired miR-142 deficit sufficed in transforming CP-LSCs into BC-LSCs, via enhancement of bioenergetic oxidative metabolism in absence of any additional gene mutations, and likely represent a novel therapeutic target in BC CML.

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	10a_M_CPZKO_1c.raw	Raw
	10b_M_CPZKO_1c.raw	Raw
	11a_M_CPZKO_2.raw	Raw
	11b_M_CPZKO_2.raw	Raw
	12a_M_CPZKO_3.raw	Raw