Fig 1.
T. annulata infection modulates the host cells miRNome.
A. Scatter plot illustrating the log2 fold change of the host cell’s miRNAs in TBL20, TBL3 and attenuated macrophages compared to BL20 and BL3 cells and virulent macrophages. Blue and red dots represent up- and down-regulated miRNAs, respectively (Log2FC>1). B. Semi-circular histogram representing the Fold Change values of the common DE miRNAs in TBL20 and TBL3 compared to their uninfected B cells (BL20 and BL3) and their expression in attenuated macrophages versus virulent macrophages. miRNAs are considered differentially expressed (DE) following two criteria: a) fold change (FC) greater than 2 and b) adjusted p value less than 0.05 (DESeq2) and FDR less than 0.1 (baySeq). Orange and green represent down and up-regulated miRNAs, respectively. The miRNA of interest, miR-126-5p, is framed in blue. C. Left. qRT-PCR confirmation of the sequencing results in Theileria-infected BL20 lymphocytes. Right. qRT-PCR confirmation of miR-126-5p and miR-126-3p levels in Theileria-infected macrophages. D. Left. qRT-PCR confirmation of the relative expression of miR-126-5p in TBL20 compared to BL20 B lymphocytes. Middle. qRT-PCR confirmation of the cellular levels of miR-126-5p following transfection of virulent macrophages with inhibitor (Vi) and attenuated macrophages with mimic sequences (Am). Right. qRT-PCR confirmation of the cellular levels of miR-126-5p in TBL20 following transfection with mimic (TBL20m) or inhibitor (TBL20i) sequences. The error bars show SD values from 3 biological replicates.
Fig 2.
Grb2 recruits PTP1B to AGO2 ablating its tyrosine phosphorylation rendering it permissive for miR-126-5p loading.
A. Relative expression of pre-miR-126 in virulent (V) and attenuated (A) Theileria-infected macrophages. B. Immunoprecipitation analyses with anti-AGO2 and anti-PTP1B antibodies using whole cell lysates derived from virulent (V) and attenuated (A) Theileria-infected macrophages. Top panel shows western blot of the AGO2 precipitate probed with anti-AGO2, anti-phospho-Tyr and PTP1B antibodies. Middle panel shows western blot of the PTP1 B precipitate probed with anti-AGO2 and anti-PTP1B antibodies. Bottom panel shows pull-down assay performed with GST-Grb2 beads and cell extracts containing endogenous AGO2 expressed by Theileria-infected macrophages. Top row shows AGO2 is bound to GST-Grb2 in virulent (V) macrophages, but in attenuated (A) macrophages the small amount of AGO2 bound to Grb2 is below the limit of detection and bottom row shows PTP1B is bound to GST-Grb2 in virulent macrophages. Anti-GST antibodies indicate the amount of Grb2 precipitated. C. Western blot analysis of total cell extracts used for immunoprecipitations probed with anti-PTP1B, anti-AGO2 and anti-actin antibodies, the latter used as a loading control. D. Relative expression of miR-126-5p in AGO2 precipitates of virulent (V) and attenuated (A) Theileria-infected macrophages. All experiments were done independently (n = 3). The error bars show SEM values from 3 biological replicates. ** p value < 0.005; *** p value < 0.001.
Fig 3.
Expression levels of miR-126-5p and its target genes dlk-1 and jip-2.
A. Expression levels of the established miR-126-5p-target dlk1 monitored as a positive control. Left. Relative expression level of dlk1 in virulent macrophages (V) compared to virulent macrophages transfected with inhibitor of miR-126 (Vi) and attenuated macrophages (A). Right. Protein level of DLK1 in TBL20 cells compared to BL20 cells and TBL20 cells transfected with miR-126-5p inhibitor (TBL20i) B. Left & right. Relative expression levels of JIP-2 in virulent macrophages (V) compared to virulent macrophages transfected with inhibitor of miR-126-5p (Vi) and attenuated macrophages (A) and attenuated macrophages transfected with the mimic of miR-126-5p (Am). The error bars show SEM values from 3 biological replicates. * p value < 0.05, ** p value < 0.01, *** p value < 0.001.
Fig 4.
miR-126-5p expression regulates the level of JIP-2 in Theileria-infected macrophages.
A. Immunoprecipitation analyses with anti-JIP-2 antibodies using whole cell lysates derived from virulent (V) Theileria-infected macrophages transfected or not with the inhibitor of miR-126-5p (Vi). The top panel (IP-JIP-2) shows western blot of the precipitate probed with an anti-DLK1 antibody, as a positive control. The lower panel shows western blot analysis of total cell extracts used for both immunoprecipitations probed with anti-JIP-2, anti-Dlk1 and anti-actin antibodies, the latter used as a loading control. B. Immunoprecipitation analyses with anti-JIP-2 using whole cell lysates derived from virulent (V) and attenuated (A) Theileria-infected macrophages transfected or not with a miR-126-5p inhibitor (Vi), mimic (Am) and an irrelevant miR control (Ac). Right and middle upper panel (IP-JIP-2) shows western blot of the precipitate probed with an anti-JIP-2 anti-JNK antibodies. Inhibition of miR-126-5p in virulent macrophages (Vi) increased the formation of the JIP-2/JNK complex, whereas stimulation of miR-126-5p in attenuated macrophages (Am) decreased complex formation. Lower panel shows western blot analysis of total cell extracts used for immunoprecipitations probed with anti-JIP-2, anti-JNK and anti-GAPDH antibodies. In panels A and B IgG represents immunoprecipitation with an irrelevant antibody (IgG). C. Relative expression of c-jun in virulent and attenuated Theileria-infected macrophages before and after transfection with the miR-126-5p inhibitor (Vi), mimic (Am) and an irrelevant control miR (Vc & Ac, NCSTUD002). The error bars show SD values from 3 biological replicates. * p value < 0.05, *** p value < 0.001 and ### p value <0.001 compared to A.
Fig 5.
miR-126-5p regulates the JNK phosphorylation of c-Jun by modulating JIP-2 levels.
A. Left. Immunofluorescence images obtained with anti-phospho-Ser73 c-Jun antibody using virulent (V) and attenuated (A) macrophages transfected or not with the inhibitor (Vi) and mimic of miR-126-5p (Am). Overexpression of miR-126-5p mimic in attenuated macrophages (Am) increased phospho-Ser73-c-Jun staining (green), whereas its inhibition (Vi) in virulent macrophages abolished phospho-Ser73-c-Jun. No fluorescence was observed with Alexa-labelled secondary antibody (Vc). Scale bar is equivalent to 10μ meters. Right. Percentage of corrected total cell fluorescence due to phospho-Ser73-c-Jun staining based on 35-independent cell images. B. Western blot of JNK isoforms (1 and 2) in nuclear and cytoplasmic extracts. JIP-2-mediated cytosolic retention of JNK1 and decreased therefore nuclear JNK1 in attenuated macrophages. Actin was used as a loading control for cytosolic extracts and histone H3 levels for loading control of nuclear extracts. All experiments were done independently (n = 3). The error bars show SEM values from 3 biological replicates ** p value < 0.01, *** p value < 0.001.
Fig 6.
miR-126-5p regulates the JNK1>AP-1 pathway by modulating JIP-2 levels that impact on matrigel traversal of infected leukocytes.
A. AP-1-(3X-TRE)-driven luciferase activity in virulent macrophages is decreased upon inhibition of miR-126-5p (Vi). When attenuated macrophages are transfected with a miR-126-5p mimic (Am), AP-1-driven luciferase activity increased. B. Left. Relative expression of mmp9 in infected macrophages (V and A) before and after transfection with miR126-5p inhibitor (Vi), mimic (Am) and an irrelevant miRNA control (Vc, NCSTUD002). Right. Relative expression of mmp9 in BL20/TBL20 B cells before and after transfection with miR126-5p inhibitor (TBL20i). C. Matrigel traversal of virulent Theileria-transformed macrophages. Inhibition of miR-126-5p expression in virulent macrophages (Vi) decreased matrigel traversal, whereas treatment with mimic (Vm) increased matrigel traversal. The reduced traversal capacity of attenuated macrophages is shown (A). All experiments were done independently (n = 3). The error bars show SEM values from 3 biological replicates. * p value < 0.05, ** p value < 0.01, *** p value < 0.001.
Fig 7.
Model proposing how Grb2 recruits PTP1B to AGO2 decreasing its tyrosine phosphorylation leading to loading and protection from degradation of miR-126-5p.
Non-degraded miR-126-5p ablates JIP-2 and DLK-1 and releases JNK to translocate to the nucleus and phosphorylate c-Jun. Phospho-c-Jun activates AP-1-driven gene transcription that underpins heightened invasiveness of Theileria-transformed virulent macrophages. In this model miR-126-3p is not loaded onto AGO2 and thus is not protected from degradation, explaining the low levels of miR-126-3p detected in virulent and attenuated macrophages.