Article Figures & Data
Figures
- Figure S1. LPS stimulated hepatocytes export out miR-122 through extracellular vesicles (EVs).
(A) Schematic representation of LPS stimulation of Huh7 cells. LPS stimulation of 100 and 500 ng/ml were given to Huh7 cells for 24 h. (B) Relative level of cellular miR-122 in LPS stimulated Huh7 cells, U6 snRNA used as control (n = 6 independent experiments; P-values = 0.0092, 0.0070, respectively). Values for untreated cells were set as unit. (C) Immunoblots for ERK1/2, P-ERK1/2, p38, P-p38, and HuR were checked in cellular lysates of LPS stimulated Huh7 cells. (C) β-Actin was used as loading control (C, upper panel). (C) Quantification of relative level of P-p38 normalized with β-Actin (n = 2 independent experiments) (C, lower panel). (D) Immunoblots for confirming the presence of EV marker proteins Alix and Flotillin-1 in EVs isolated from control and LPS stimulated Huh7 cells. (E) Quantification of miR-122 levels in EVs isolated from LPS stimulated Huh7 cells. EV protein markers were used for normalization of miR-122 levels (n ≥ 3 independent experiments; P-values = 0.0809, 0.1085, respectively). Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, **P < 0.01. P-values were calculated by two-tailed paired t test unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
- Figure 1. Hepatic miR-122 acts as an immunostimulant for mammalian macrophage and Ld restricts miR-122 entry in infected macrophage.
(A) Diagrammatic depiction of experiments done with Leishmania donovani (Ld)–infected or uninfected RAW264.7 cells treated with miR-122–containing extracellular vesicles (EVs). The macrophage cells were either infected or noninfected with Ld for 6 h after which both the groups were either treated with miR-122–containing EVs or left untreated. (B) Western blot analysis of RAW264.7 cells to show the infection related up-regulation of Ucp2 and down-regulation of HRS protein expression in RAW264.7 cells (left panel). Graphical representation of densitometric analysis of Ucp2 Western blot (right panel) (n ≥ 3 independent experiments, unpaired t test, P = 0.2139). Values for uninfected and untreated cells were set as unit. (C) Effect of miR-122 overexpression on cytokine expression and nitric oxide generation in RAW264.7 cells. The RAW264.7 macrophage cells were either kept as control, transfected with miR-122 expression plasmid pmiR-122 or with miR-146a expression plasmid pmiR-146a or treated with LPS (as positive control) to determine the mRNA level of TNF-α (C, left panel, n = 3 independent experiments; P = 0.0361, 0.0084) and iNOS (C, middle right panel, n = 3 independent experiments; P = 0.0562, 0.0002) from cellular RNA. Protein level of TNF-α (C, middle left panel, n = 3 independent experiments; unpaired t test, P = 0.0006) and nitric oxide level (right panel, n = 3 independent experiments; unpaired t test, P ≤ 0.0001, 0.0405, <0.0001) was also measured from culture supernatant. Quantification of TNF-α mRNA levels by qRT-PCR was done for the conditions described above. 18s RNA or GAPDH mRNA was used as endogenous control for qRT-PCR. Values for control plasmid transfected untreated cells were considered as unit for qRT-PCR (control). (D) Effect of miR-122 expression on Ld infection. This panel shows the schematic model for RAW264.7 cells transfected with miR-122 expression plasmid (pmiR-122) followed by L. donovani infection. (E) Comparison of the proinflammatory TNF-α, and anti-inflammatory IL-10 cytokine expression in pmiR-122 transfected or pCIneo control vector transfected RAW264.7 cells followed by infection with Ld. An increase in the proinflammatory cytokine TNF-α (P = 0.0180) and a decrease in the anti-inflammatory cytokine IL-10 (P = 0.0422) were observed in the presence of miR-122 (n = 3 independent experiments).18s rRNA was used as endogenous control. Values for pCIneo control transfected and infected RAW264.7 cells were considered as unit. (F) Microscopic analysis of Ld infection in RAW264.7 cells treated or untreated with miR-122–containing EVs. Cells were then visualized under the confocal microscope. The Leishmania protein GP63 imaged with indirect fluorescence (red) and cells with internalized parasites were counted. Scale bar 8 μm. Marked areas are zoomed for 5×. (G) Effect of miR-122–containing EVs on Ld infection. Graphical representation of the percent of RAW264.7 cells infected with L. donovani as observed microscopically (n = 132, number of cells; P = 0.0038, unpaired t test). (H) Quantification of miRNA levels upon Ld infection followed by EV treatment in RAW264.7 cells. Real-time PCR showed no increase in miR-122 levels in infected macrophages incubated with miR-122 positive EVs (top left panel; P = 0.0215, 0.3211, 0.3808). miR-155 levels were also quantified under similar conditions (bottom left panel; P = 0.0024, 0.0736, 0.1368) (n = 4 independent experiments). U6 was used as endogenous control. Relative levels of cytokine mRNA were also quantified. The proinflammatory cytokine, TNF-α (top right panel; P = 0.0142, <0.0001, 0.0013) and IL-1β (bottom right panel; P = 0.0438, <0.0001, 0.0366) did not increase in presence of the parasite followed by EV treatment (n = 4 independent experiments). 18s rRNA was used as endogenous control. Values for uninfected cells were set as unit. (I) Quantification of iNOS mRNA level and Nitric oxide (NO) generation after miR-122–containing EV treatment of RAW264.7 cells. Nitric oxide level was determined from culture supernatant of EV-treated RAW264.7 cells (upper panel, n = 3 independent experiments, unpaired t test; P = 0.3878). qRT-PCR analysis was used to determine the relative level of iNOS mRNA in recipient RAW264.7 cells (lower panel, n = 3 independent experiments; P = 0.1275). GAPDH mRNA was used as endogenous control. Values for control EV-treated cells were considered as unit for qRT-PCR. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
Source data are available for this figure.
Source Data for Figure 1[LSA-2021-01229_SdataF1.pdf]
- Figure 2. miRNA uptake is prevented in Ld-infected primary macrophages and Kupffer cells.
(A) A model to show the effect of Leishmania infection on uptake of miR-122–containing extracellular vesicles (EVs) in primary peritoneal macrophages. Macrophages were either infected or noninfected with Ld followed by miR-122–containing EV treatment. The EVs were obtained from miR-122 expressing murine hepatic cell HePa 1–6. (B) Relative levels of internalized miR-122 in infected or noninfected primary macrophages (n = 3 independent experiments; 0.0379, 0.0033, 0.2726, Unpaired t test P = 0.0227). U6 was used as endogenous control and values for uninfected control cells were considered as unit. (C) Schematic representation of Ld infection of mice followed by miR-122–containing EV treatment. Mice were infected with Ld for 30 d followed by tail vein injection of miR-122–containing EVs. After 24 h of treatment, mice were euthanized and Kupffer cells were isolated to quantify the miR-122 content. (D) Characterisation of isolated hepatocytes and Kupffer cells. Quantification of hepatocyte specific albumin mRNA in hepatocytes and Kupffer cells to show their low levels in Kupffer cell isolate (left panel; P = 0.143, n = 4 number of mice). Relative levels of macrophage specific C-type Lectin Domain Family 4, Member F (Clec4f) mRNA between hepatocytes and Kupffer cells showing high levels in Kupffer cells (right panel; P = 0.0378, n = 4 number of mice). GAPDH was used as endogenous control. Values for hepatocytes were considered as unit. (E) Comparison of normalized Ct values of Kinetoplast DNA in infected or uninfected Kupffer cells isolated from animals untreated or treated with EVs containing miR-122 to determine the parasitic load in Kupffer cells (n = 3 number of mice; P = 0.0009, 0.0005, unpaired t test). GAPDH was used as endogenous control. (F) Mean Ct values of internalized miR-122 in uninfected Kupffer cells either untreated or treated with miR-122–containing EVs (n = 3 number of mice; P = 0.0049, unpaired t test). (G) Mean Ct values of TNF-α mRNA in uninfected Kupffer cells upon treatment with miR-122–EVs compared with untreated Kupffer cells (n ≥ 2; P = 0.0444, unpaired t test). (H) The internalization of EV-derived miR-122 declined in the presence of Leishmania infection. Real-time PCR revealed the relative levels of miR-122 in uninfected or infected Kupffer cells isolated from animals treated with miR-122–containing EVs (n = 3 number of mice; P = 0.0162). U6 was used as endogenous control and values for uninfected miR-122 EV-treated cells were considered as unit. (I) Relative levels of proinflammatory cytokine TNF-α in Leishmania infected or uninfected miR-122–EV-treated animals’ Kupffer cells (n = 3 number of mice; P < 0.0001). GAPDH mRNA was used as endogenous control and values for uninfected miR-122 EV-treated set were considered as unit. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise.
- Figure S2. Effect of Ld infection on the endosomes of RAW264.7 cells.
(A) Effect of Leishmania infection on the endosome number of RAW macrophages. Microscopic images to show the endosomes (YFP-Endo, green) and Leishmania parasite (GP63 protein, red). Scale bar 3 μm for uninfected and 5 μm for infected RAW cells expressing YFP-Endo protein. (B) Graphical representation of the number of endosomes in uninfected or infected macrophages (n = 15; unpaired t test, P = 0.1045). (C) Western blots to show the levels of different endocytic pathway component proteins (Dynamin 2, Rab5a, and RILP) under different conditions of infection and extracellular vesicle-treatment in RAW264.7 cells. β-Actin used as loading control. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant. P-values were calculated by two-tailed paired t test. Positions of molecular weight markers are marked and shown with the respective Western blots.
Source data are available for this figure.
Source Data for Figure S2[LSA-2021-01229_SdataFS2.pdf]
- Figure 3. Mitochondrial depolarization and ER detethering by Ld prevents internalization and activity of extracellular vesicle (EV)-derived miRNAs in recipient cells.
(A) Structure of mitochondria (green) in Mito-GFP–expressing HeLa cells transfected with FH-Ucp2 (red) or control plasmid. Scale bar 5 μm. (B) Graphical representation of the internalization of CD63-GFP–positive EVs in recipient HeLa cells transfected either with control or FH-Ucp2–expression plasmids (n = 18, number of cells; P < 0.0001, unpaired t test). (C) Effect of FH-Ucp2 expression on uptake of miR-122. Relative level of internalized EV-derived miR-122 in recipient HeLa cells transfected either with control or FH-Ucp2 expression plasmids (left panel; n = 3 independent experiments; P < 0.0001). U6 was used as endogenous control and values for control plasmid transfected and miR-122 EV-treated cells was considered as unit. Right panel shows the Western blot for HA. β-Actin was used as loading control. (D) Luciferase assay to show the repression levels of RL-perf-miR-122 in recipient cells transfected with pCIneo control or FH-Ucp2–expression plasmids and treated with miR-122–containing EVs. The repression levels were calculated as a ratio of firefly normalized RL-Control value to firefly normalized RL-perf-miR-122 value (n = 3 independent experiments; 0.0117, 0.0987, unpaired t test = 0.0013). (E) Relative level of internalized miR-122 (left panel; P = 0.0012) and endogenous miR-16 levels (right panel; P = 0.0316) in Mfn2 (Mitofusin2) wild-type and knockout MEF cells treated with miR-122–containing EVs. Quantification was done by qRT-PCR and U6 levels were used as normalizing control (n = 3 independent experiments). Values for Mfn2 wild-type cells were set as unit. (F) FH-Ago2 associated miR-122 levels in Mfn2 wild-type and knockout MEF cells (transfected with FH-Ago2) co-cultured with miR-122 expressing HeLa cells. Immunoprecipitated Ago2 levels were used for normalization of associated miR-122 (n = 3 independent experiments; P = 0.0077). Values for Mfn2 wild-type cells was considered as unit. (G) Rescue of EV-miR-122 internalization in Leishmania donovani infected RAW264.7 cells in presence of genipin, the inhibitor of Ucp2. RAW264.7 cells infected with Ld were treated with genipin (100 μM after 4 h of infection) followed by addition of miR-122 positive EVs. Relative levels of miR-122 (top panel; P = 0.0311) and TNF-α mRNA (bottom panel; P = 0.0968) increased in infected RAW264.7 cells treated with genipin (n = 3 independent experiments). U6 levels were used as control for normalization of miRNA and 18s rRNA was used normalizing levels for mRNA. Values for infected and genipin untreated set was considered as unit. (H) Diagrammatic representation of the EV-mediated crosstalk between hepatocytes and macrophages. Hepatocytes release miR-122–containing EVs which can be transferred to macrophages that causes production of proinflammatory cytokines and can prevent Ld infection (red arrow). Inversely, Ld-infected macrophages are unable to take up the miR-122–containing EVs due to up-regulation of Ucp2 protein in cells and are tuned to have high production of anti-inflammatory cytokines for sustained parasitic infection (green arrow). Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
Source data are available for this figure.
Source Data for Figure 3[LSA-2021-01229_SdataF3.pdf]
- Figure S3. Effect of FH-Ucp2 expression and Leishmania infection on ER–mitochondria interaction in HeLa and RAW264.7 cells.
(A) Microscopy of the ER (red) and mitochondria (green) interaction in Mito-GFP expressing HeLa cells untransfected or transfected with FH-Ucp2. Scale bar 10 and 5 μm for control zoomed surface image and 3 μm for FH-Ucp2 expressed zoomed surface image. (B) Microscopy of RAW264.7 cells to show the ER (red) and mitochondria (green) interaction in cells uninfected or infected with Ld. The Leishmanial protein GP63 was depicted in blue to show the parasite. Scale bar 5 and 2 μm for control zoomed surface image and 1 μm for Ld-infected zoomed surface image. (C) Relative level of miR-122 was determined by qRT-PCR in recipient RAW264.7 cells after 3 h pre-treatment with either oligomycin (2.5 μM) or FCCP (0.5 μM) followed by 16 h treatment with miR-122–containing extracellular vesicles derived from pmiR-122 transfected Huh7 cells (n = 3 independent experiments, P = 0.0575 and n = 3 independent experiments, P = 0.8436, respectively). Ethanol (ETOH) or DMSO treated cells were used as controls for oligomycin or FCCP, respectively. U6 used as endogenous controls. (D) Relative level of miR-122 in FH-Ucp2 and pmiR-122 overexpressed RAW264.7 cells (D, left panel, n = 3 independent experiments, P = 0.0396 and right panel, n = 3 independent experiments, P = 0.2259, respectively). U6 used as endogenous control and values for pCIneo transfected cells were set as unit. (E) Quantification of ATP content in pmiR-122 overexpressed RAW264.7 cells (n = 5 independent experiments, unpaired t test, P = 0.0101). Emitted light intensities from samples in a luciferase-based ATP measurement system represents concentration of ATP in samples used for comparison. (F) Changes in mean fluorescence intensities obtained from RAW264.7 cell stained for MitoTracker red and analysed in a FACS based mitochondrial depolarization measurement assay. RAW264.7 cells transfected either with pCIneo control vector or miR-122 expressing plasmids were used for analysis (n = 3 independent experiments, unpaired t test, P = 0.1789). (G) Relative protein level of Ucp2 in pCIneo, pmiR-122 and FH-Ucp2 transfected RAW264.7 cells after normalization (G, upper panel, n = 3 independent experiments, P = 0.0116, 0.1452). Values for pCIneo transfected cells were set as unit. Western blot for Ucp2 and HA was given in lower panel. β-Actin was used as loading control and used for normalization. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *P < 0.05. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
- Figure 4. Leishmania donovani (Ld) infection triggers extracellular export of miRNAs from infected macrophage cells.
(A) Schematic representation of the experiment. After 24 h of Ld infection, the host macrophage and extracellular vesicles (EVs) isolated from culture supernatant were analysed for cellular and EV-associated protein and RNA analysis. (B) Ld infection altered cellular protein levels. Western blot images showed protein levels after 24 h of infection of macrophages. β-Actin was used as loading control. (C, D, E) Characterisation and quantification of EVs released from control and Ld-infected macrophages. Nanoparticle Tracking Analysis (NTA) of EVs released from control (C, left panel) and infected cell (C, right panel). (D) Western blot images showed EV marker proteins in control and infected cell released EVs. (E) Number and size of EVs released by control and infected cells were quantified by NTA analysis (E, left panel, P = 0.0008 and right panel, P = 0.0677, n = 3 independent experiments, respectively, unpaired t test). (F, G) miR-146a level increases and the miRNA gets exported from Ld-infected macrophage cells. qRT-PCR based relative quantification of cellular miR-155 (F, left panel, P = 0.0817, n = 3 independent experiments) and miR-146a (F, right panel, P = 0.0146, n = 4 independent experiments) after 24-h infection of RAW264.7 cells. U6 was used as endogenous control. miRNA levels of infected cells were normalized against noninfected controls. qRT-PCR data represented the relative level of miR-146a and miR-155 in EVs from RAW264.7 cells after infection. EV marker protein Alix was used for normalization of EV-associated miR-146a level (G, P = 0.0229, n = 4 independent experiments). Values for uninfected cell–derived EVs were set as unit. (H) Ld infection triggers the extracellular export of miRNAs from mouse peritoneal macrophages. miR-146a level was also estimated in EV-derived from mouse peritoneal macrophage (H, upper panel, P = 0.0006, n = 5 independent experiments, unpaired t test) and Western blot data also showed EV marker proteins (H, lower panel). (I) Average Ct value of miR-21 (I, left panel, P = 0.0011, n = 3 independent experiments, unpaired t test), miR-125b (I, middle panel, P = 0.2654, n = 3 independent experiments, unpaired t test), and miR-16 (I, right panel, P = 0.0794, n = 3 independent experiments, unpaired t test) in EVs released from Ld-infected and noninfected RAW264.7 cells. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
Source data are available for this figure.
Source Data for Figure 4[LSA-2021-01229_SdataF4.pdf]
- Figure S4. Proteomics profile of control and infected cell derived extracellular vesicles (EVs) were isolated from control and infected cells and a pooled sample of two 90 mm tissue culture dishes were used for proteomics analysis.
Ven diagram showed 20 common proteins present in both control and infected cell–derived EVs. The list showed the types of proteins found specifically in both types of EVs along with common proteins.
- Figure S5. Subcellular sequestration of cytokine mRNAs, miRNAs and their targets in Ld-infected macrophage cells.
(A, B, C, D) Polysomal sequestration of cytokine mRNAs, miRNAs and their targets after 24 h of Ld infection. (A) Relative levels of miR-155 and miR-146a in polysomes were measured by qRT-PCR (A, left panel, P = 0.0775, n = 5 independent experiments and right panel, P = 0.0077, n = 3 independent experiments respectively). (B) Relative levels of polysomal MyD88 and TRAF6 mRNA levels were determined by qRT-PCR (B, left panel, P = 0.7312, n = 3 independent experiments and right panel, P = 0.0802, n = 3 independent experiments, respectively). (C) Relative levels of polysome associated cytokine mRNA levels of TNF-α (C, left panel, P = 0.0783, n = 4 independent experiments), IL-10 (C, right panel, P = 0.3740, n = 3 independent experiments) were measured by qRT-PCR in control and infected cells. For mRNA, 18s rRNA or GAPDH was used for normalization and for miRNA, polysomal marker S3 or L7a was used for normalization. Values for uninfected control cells were set as units. (D) Polysomal proteins (L7a and Ago2) were determined by Western blot. (E, F, G) Subcellular localization of miR-146a and miR-155 in control and infected RAW264.7 cells. (E) Western blot analysis revealed the marker proteins of MVB and ER. (F, G) Relative miR-155 and miR-146a levels were determined by qRT-PCR in MVB and ER fractions of control and infected cells (F, n ≥ 3 independent experiments, P = 0.0234, unpaired t test and G, n ≥ 3 independent experiments, P = 0.0514, unpaired t test). miRNA levels were normalized by corresponding marker proteins. Values of control cells were set as units. (H) Levels of HuR change with time of infection in Ld-infected cells. Cell lysates were Western blotted for HuR and β-Actin was used as loading control. (I) Increased association of miR-146a and miR-155 with HuR in LPS-treated cells. RAW264.7 cells were treated with LPS (100 ng/ml) for 8 h and HuR was immunoprecipitated. (I) The amount of miRNAs associated with HuR in control and LPS-treated cells were measured by real-time PCR (n = 3 independent experiments; P-values: 0.0658, 0.0615, respectively) (I, upper panel). The amount of HuR-associated miRNA were normalized against the amount of HuR immunoprecipitated (n = 3 independent experiments) (I, lower panel). Values of untreated cells were considered as unit. (J, K) Comparison of association of miR-146a and miR-155 with HuR and extracellular vesicles secreted from the pHA-HuR expressing RAW264.7 cells. (J) The average Ct values of specific miRNAs were plotted for cellular and HuR immunoprecipitated material from LPS stimulated cell equivalent amounts of samples (n = 3 independent experiments). (K) The average Ct values of the cellular and extracellular vesicle–associated miRNAs in pHA-HuR transfected RAW264.7 cells were plotted (n = 3 independent experiments). Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *P < 0.05, **P < 0.01. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
Source data are available for this figure.
Source Data for Figure S5[LSA-2021-01229_SdataFS5.pdf]
- Figure 5. Extracellular vesicle (EV)-mediated transfer of Ld-infected cell derived miR-146a reduces miR-122 level in hepatic cells.
(A, B) Effect of control and Ld-infected cell–derived EVs on naïve hepatocytes. (A) A schematic representation of the experiment (A). (B) Relative levels of miR-146a (B, left panel, P = 0.0329, n = 3 independent experiments), miR-122 (B, middle left panel, P = 0.0364, n = 4 independent experiments), Pre-miR-122 (B, middle right panel, P = 0.0993, n = 3 independent experiments), miR-16 (B, right panel, unpaired t test P = 0.2074, n = 3 independent experiments) were measured by qRT-PCR in recipient hepatic Huh7 cells after 24 h of control and infected cell–derived EV treatment. U6 was used as endogenous control for miRNA normalization and for Pre-miR-122 level, GAPDH was used as endogenous control. Values for control EV-treated cells were used for normalization of infected cell–EV-treated cells. (C, D) Effect of pmiR-146a–expressing macrophage derived-EV treatment on recipient hepatocytes. (C) A schematic representation of the experiment has been described in panel. (D) Relative levels of miR-146a (D, left panel, P = 0.0213, n = 3 independent experiments), miR-122 (D, middle panel, P = 0.0036, n = 3 independent experiments) and Pre-miR-122 (D, right panel, P = 0.0307, n = 3 independent experiments) were measured by qRT-PCR in recipient Huh7 cells after pCIneo and pmiR-146a transfected RAW264.7 cell–derived EV treatment for 24 h. U6 was used as endogenous control for cellular miRNA level normalization. Values for pCIneo-transfected cell–derived EV-treated cell were set as unit. For Pre-miR-122 level, GAPDH was used as endogenous control. (E) Effect of pmiR-146a overexpression on miR-122 level in hepatocytes. Relative levels of miR-146a (E, left panel, P = 0.2251, n = 3 independent experiments), miR-122 (E, middle panel, P = 0.0003, n = 4 independent experiments) and Pre-miR-122 (E, right panel, P = 0.0279, n = 4 independent experiments) were measured by qRT-PCR in Huh7 cells after pCIneo and pmiR-146a expression in hepatocytes. U6 was used as endogenous control for miRNA normalization. pCIneo transfected cells was used for normalization of pmiR-146a transfected cells. For Pre-miR-122 level, GAPDH was used as endogenous control. (F, G, H) Effect of EV treatment on Dicer1 level in naïve hepatocytes. (F) Level of Dicer1 was measured by Western blot in control and Ld-infected cell–EV-treated Huh7 cells. β-Actin was used as loading control (F). (G) Dicer1 level was also measured in Huh7 treated with EVs derived from pCIneo and pmiR-146a transfected RAW264.7 cells. β-actin was used as loading control. (H) Effect of pmiR-146a expression on Dicer1 in Huh7 hepatocytes was also determined. β-Actin was used as loading control. (I) Effect of pmiR-146a overexpression on signalling component proteins in hepatocytes. Levels of HuR, P-ERK1/2, P-p38 were determined by Western blot analysis in pCIneo and pmiR-146a transfected Huh7 cells. β-Actin was used as loading control. (J, K) Effect of NHA-Dicer1 expression on Dicer1 associated precursor miRNA level in cells expressing pmiR-146a. (J) Average Ct value of NHA-Dicer1 associated Pre-miR-146a (J, left panel, P = 0.0005, n = 3 independent experiments, unpaired t test) and Pre-miR-122 level (J, right panel, P = 0.2630, n = 3 independent experiments, unpaired t test) were determined by Real-time PCR after HA-Dicer1 was immunoprecipitated from pCIneo and pmiR-146a transfected Huh7. (K) NHA-Dicer1 was immunoprecipitated using anti-HA antibody and was detected in immunoprecipitated and input samples using anti-HA antibody. HC, heavy chain. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
Source data are available for this figure.
Source Data for Figure 5[LSA-2021-01229_SdataF5.pdf]
- Figure S6. miR-146a does not change extracellular vesicle (EV) number or concentration in macrophage cells.
(A) Nanoparticle tracking analysis of EVs isolated from pCIneo (A, left panel) or pmiR-146a (A, right panel) transfected RAW264.7 cells. The size and concentration of the respective EVs were compared (n = 2 independent experiments). PBS was used as blank for nanoparticle tracking analysis. (B) Ct value of EV-associated miR-146a isolated from pCIneo or pmiR-146a transfected RAW264.7 cells (n = 3). (C) Immunoblotting was performed for specific marker proteins which are known to be present and absent in EVs. Both cellular and EV isolated from pCIneo and pmiR-146a transfected RAW264.7 cells were analysed for the respective marker proteins. β-Actin was used as loading control for the cellular samples. (D) Effect of miR-122 or miR-146a overexpression on IL-1β expression in RAW264.7 cells. Protein level of IL-1β from cell lysate of pCIneo, pmiR-122, and pmiR-146a transfected RAW264.7 cells was determined by ELISA (n = 3 independent experiments, unpaired t test, P = 0.2075, 0.0007). Data information: In all experimental data, error bars are represented as mean ± SEM. Positions of molecular weight markers are marked and shown with the respective Western blots.
- Figure 6. Extracellular vesicle (EV)–mediated transfer of miR-146a from infected macrophage cells promotes anti-inflammatory response in naïve recipient macrophages.
(A, B) Effect of EV treatment on LPS induced activation of recipient macrophages. (A) A schematic representation of the experiment has been shown in the left. (B) Relative cellular levels of TNF-α (B, left panel, P = 0.0224, n = 6 independent experiments), IL-1β (B, middle left panel, P = 0.0280, n = 5 independent experiments) mRNAs were measured by qRT-PCR from Infected cell–EV-treated macrophages after LPS treatment. TNF-α and IL-1β (B, middle right, P = 0.2487, n = 6 independent experiments, unpaired t test and right panel, P = 0.1133, n = 6 independent experiments, unpaired t test, respectively) were measured by qRT-PCR from infected cell derived Microvesicle (MV)–treated macrophages followed by 4 h of LPS (1 ng/ml) treatment. 18s rRNA was used as endogenous control. Values in the noninfected cell EV treated set were used as units. (C, D, E, F) Effect of infected cell–derived EV treatment on recipient macrophages. (C) A schematic representation of the experiment has been shown (C). (D) Relative levels of miR-146a and iNOS mRNA in recipient macrophage after 24 h of EV treatment were measured by qRT-PCR and miRNA and mRNA levels in noninfected cell EV-treated cell were set as unit. U6 and GAPDH was used as endogenous control for miRNA and mRNA, respectively (D, left panel P = 0.0184, n = 4 independent experiments and right panel, P = 0.0035, n = 5 independent experiments, respectively). (E) Levels of Dicer1, HuR, P-ERK1/2, and P-p38 were measured by Western blot analysis in infected cell–derived EV-treated RAW264.7 cells. β-Actin was used as loading control. (F) Relative levels of IL-1β (F, left panel, P = 0.0967, n = 3 independent experiments) and IL-10 (F, right panel, P = 0.0357, n = 3 independent experiments) were measured by qRT-PCR in recipient RAW264.7 cells after 24 h of EV treatment. GAPDH was used as endogenous control and values for noninfected cell EV-treated cells were set as unit. (G, H) Effect of EV-mediated transfer of miR-146a on recipient macrophage. (G) A schematic diagram has been given. (H) Relative levels of cytokine mRNAs; IL-1β, IL-10 and iNOS (H, upper panel, P = 0.2131, n = 3 independent experiments, H, middle panel, P = 0.0406, n = 4 independent experiments and H, lower panel, P = 0.0662, n = 3 independent experiments, respectively). GAPDH was used as endogenous control and values for pCIneo control EV-treated cells were set as units. (I) Effect of Ld infection on cytokine mRNA levels in pmiR-146a overexpressed macrophages. A schematic diagram of the experiment has been shown (I, upper panel). Relative cytokine mRNA level of TNF-α (I, left panel, P = 0.0138, P = 0.1740, P = 0.7287, n = 3 independent experiments), IL-1β (I, middle panel, P = 0.0183, P = 0.0684, P = 0.4727, n = 3 independent experiments), and IL-10 (I, right panel, P = 0.1491, P = 0.2417, P = 0.7457, n = 3 independent experiments) were measured by qRT-PCR from pCIneo and pmiR-146a transfected RAW264.7 cells after 24 h of Ld infection or no infection. GAPDH was used as endogenous control and values for pCIneo control cells were set as units (I, unpaired t test was performed for comparison between pmiR-146a–expressing noninfected control and infected set). (J) Effect of control and infected cell derived-EV treatment on recipient macrophages transfected with control or miR-146a inhibitor oligos. A schematic diagram has been given (J, left panel). Relative mRNA level of IL-10 was measured by qRT-PCR from miR-146a inhibitor transfected and EV-treated RAW264.7 cells (J, right panel, paired t test, P = 0.0149,0.3208, unpaired t test was performed for comparison between miR-146a inhibitor transfected control EV and infected cell EV-treated sets, P = 0.0456, n = 3 independent experiments, respectively). GAPDH was used as endogenous control and values for negative control inhibitor transfected cells were set as units. (K) Effect of LPS treatment on pmiR-146a–expressing macrophages. Relative level of IL-10 was measured by qRT-PCR from control or pmiR-146a–expressing cells with or without LPS exposure (1 ng/ml) (K. paired t test, P = 0.0984, 0.0921, unpaired t test was performed for comparison between LPS-treated sets, P = 0.0132, n = 3 independent experiments). GAPDH was used as endogenous control and values for untreated control cells were set as units. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *P < 0.05, **P < 0.01, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
Source data are available for this figure.
Source Data for Figure 6[LSA-2021-01229_SdataF6.pdf]
- Figure S7. Effect of parasite-derived extracellular vesicles (EVs), SLA, and LPG on cytokine mRNA, miRNA level, and signalling components.
(A, B, C) Effect of parasite secreted EVs on cellular proteins and cytokine levels in naïve RAW264.7 cells. Protein levels of Dicer1, HRS, Ago2, P-p38, P-ERK1/2, P-p65, and Rab5a were determined by Western blot of control and Ld EV-treated macrophages. (A) β-Actin was used as loading control. (B) Relative level of P-p38 protein in recipient macrophages treated with control and Ld-infected cell-derived EVs was determined by Western blot and quantified against β-Actin (B, left panel, P = 0.1041, n = 3 independent experiments). Relative level of iNOS mRNA in recipient macrophages treated with control and parasite EVs was determined by qRT-PCR (B, right panel, n = 3 independent experiments, P = 0.0003). Values of control EV-treated cells were considered as units. (C) Relative levels of TNF-α (C, left panel, P = 0.0252 n = 4 independent experiments), IL-1β (C, middle panel, P = 0.0002 n = 4 independent experiments), and IL-10 (C, right panel, P = 0.1583, n = 3 independent experiments) mRNA were measured by qRT-PCR in recipient macrophages treated with parasite EVs and medium control EVs for 24 h. GAPDH was used as endogenous control. Values for control EV-treated cells were considered as units. (D, E) Relative levels of miR-122 in RAW264.7 cells (D, left panel, n = 3 independent experiments, P = 0.0861) and miR-146a in Huh7 cells (D, right panel, n = 3 independent experiments, P = 0.0025) were determined after medium control EV and parasite EV treatment. RNA estimation was quantified by qRT-PCR. U6 was used as endogenous control. Values for control EV-treated cells were considered as unit. Relative levels of miR-122 in RAW264.7 cells (E, upper panel, n = 3 independent experiments, P = 0.5022) and miR-146a in Huh7 cells (E, lower panel, n = 3 independent experiments, P = 0.1160) were determined after treatment with Ld-derived Soluble Leishmanial Antigen (SLA) at 10 μg/ml for 24 h, by qRT-PCR. U6 was used as endogenous control. Values for control untreated cells were considered as units. (F, G) Effect of Ld-derived LPG treatment on cellular proteins and cytokine mRNAs. Crude LPG was purified from Ld lysate and resuspended in sterile PBS before treatment. RAW264.7 cells were left untreated or treated with LPG at a dose of 1:50 (macrophage: Ld cell derived LPG) for 24 h and cellular protein levels (P-p38, p38, P-ERK1/2, and ERK-1/2) were determined by Western blots. β-Actin was used as loading control (F). Relative mRNA level of IL-1β (G, upper left panel, P = 0.8346, n = 3 independent experiments), TNF-α (G, upper middle panel, P = 0.0027, n = 3 independent experiments) and IL-10 (G, upper right panel, P = 0.4919, n = 3 independent experiments) were determined by qRT-PCR. GAPDH was used as endogenous control. Relative levels of miR-122 in RAW264.7 cells (G, lower left panel, n = 3 independent experiments, P = 0.0586) and miR-146a (G, lower right panel, n = 3 independent experiments, P = 0.1152) in Huh7 cells were determined by qRT-PCR after treatment of cells with Ld-derived LPG used at 1:50 ratio for 24 h. U6 was used as endogenous control. Values for untreated cells were considered as units. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *, ** and *** represent P-value of <0.05, <0.01 and <0.001, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
- Figure 7. Crosstalk between miRNA-exporter HuR and miR-146a determines the fate of cytokine response in macrophage cells.
(A) LPS treatment (100 ng/ml) for different time points in RAW264.7 cells. The cell lysates were immunoblotted for HuR. β-Actin was used as loading control. (B) Real-time PCR was performed for proinflammatory cytokine mRNAs (TNF-α and IL-1β) in pCIneo and pHA-HuR cells co-transfected with pre-miR control mimic. GAPDH was used for normalization (n = 5 independent experiments; P-values = 0.2048, 0.0467, respectively). Values observed in pCIneo transfected cells were considered as units for normalization of values obtained with pHA-HuR transfected cells. (C, D) Extracellular vesicles (EVs) isolated from pmiR-146a transfected RAW264.7 cells were treated to recipient RAW264.7 cells expressing pCIneo or pHA-HuR. (C) Recipient cells treated with pmiR-146a–expressing cell EVs were immunoblotted for HA and HuR. β-Actin was used as loading control. (D) Real-time PCR was performed for cytokine mRNAs (TNF-α, IL-1β, and IL-10) in pCIneo and pHA-HuR transfected recipient cells treated with pmiR-146a–EVs. GAPDH was used for normalization (n = 3 independent experiments; P-values: 0.3952, 0.0134, and 0.0352, respectively). Values obtained with pCIneo transfected and EV-treated cells were considered as units. (E, F) EVs were isolated from pmiR-146a transfected RAW264.7 cells and used to treat the recipient RAW264.7 cells depleted of HuR (siHuR transfected). (E) Recipient cells treated with pmiR-146a–EVs were immunoblotted for HuR to check for proper silencing and β-Actin was used as loading control (E). (F) Real-time PCR was performed for cytokine mRNAs (TNF-α, IL-1β and IL-10) in siCon or siHuR-containing recipient cells treated with pmiR-146a–EVs, GAPDH was used for normalization (n = 3 independent experiments; P-values: 0.0004, 0.0015, 0.2641, respectively) (F). In this particular Experiment, siCon transfected and EV-treated cells was used for normalization of values noted in siHuR-containing cells. (G, H, I, J, K) Effect of pHA-HuR expression in RAW264.7 cells transfected with pre-miR control mimic or pre-miR-146a mimic. Transfection was performed either with pCIneo (control plasmid) or pHA-HuR. (G) Immunoblotting of the cell lysates for HuR using β-Actin as loading control (G). (H) Immunoblotting of the respective cell lysates for HA to check for proper overexpression of HA-HuR, β-Actin was used as loading control. Real-time PCR was performed for TNF-α, where GAPDH has served as control. (I) Values obtained for pCIneo and pre-miR control mimic co-transfected sets were taken as units (n ≥ 5 independent experiments; P-values: 0.2048, 0.8587, 0.8620, respectively). (J) Real-time PCR was performed for IL-1β, GAPDH served as control. Values obtained for pCIneo and pre-miR control mimic co-transfected sets were taken as units (n ≥ 5 independent experiments; P-values: 0.0467, 0.3453, 0.0375, respectively). (K) Real-time PCR was performed for IL-10, where GAPDH has served as control. Values obtained for pCIneo and pre-miR control mimic co-transfected sets were taken as units (n ≥ 4 independent experiments; P-values: 0.0239, 0.2710, 0.0267, respectively). (L, M) Relative level of miR-21 in HA-HuR expressing RAW264.7 cells. Real-time PCR for miR-21 level in RAW264.7 cells expressing pHA-HuR and transfected with pre-miR-146a mimic. Values obtained for pCIneo and pre-miR mimic co-transfected sets were taken as units. U6 snRNA served as control (n = 3 independent experiments; P-value = 0.0015) (L, left panel). Real-time PCR for miR-21 level in recipient RAW264.7 cells depleted of HuR which were treated with miR-146a–containing EVs. Values obtained for siCon and miR-146a–containing EV-treated sets were taken as units. U6 snRNA served as control (n = 3 independent experiments; P-value = 0.5259) (L, middle panel). Real-time PCR of EV-associated miR-21 level from EVs of RAW264.7 cells expressing or not expressing pHA-HuR. EV marker protein Flotillin-1 was used for normalization of miR-21 level (n = 3 independent experiments; P-value = 0.4313) (L, right panel). Values obtained for pCIneo transfected cell–derived EVs was considered as unit. (M) Immunoblotting of the cell lysates with HA using β-Actin as loading control in pCIneo or pHA-HuR transfected RAW264.7 cells. (N) The proposed model depicts the role of EV-associated miRNAs in Ld infection. The left half shows that Ld infection triggers EV-mediated secretion of miR-146a from macrophage which when transfers to naïve macrophage polarizes it toward M2 phenotype because of its anti-inflammatory role whereas the right half represents what happens when miR-146a get delivered to naïve hepatocyte via EV. miR-146a reduces miR-122 level in hepatocytes. If not restricted, miR-22 as part of hepatocyte secreted EVs polarizes naive macrophage to M1 phenotype because of its proinflammatory role. Data information: In all experimental data, error bars are represented as mean ± SEM,*P < 0.05; **P < 0.01; ***P < 0.001. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
Source data are available for this figure.
Source Data for Figure 7[LSA-2021-01229_SdataF7.pdf]
- Figure 8. Effect of extracellular vesicle (EV)–associated miR-146a treatment on Ld infection.
(A, B, C, D) Infected cell–derived EV treatment decreases Ld infection in recipient cells. (A) Parasites were labelled with CFSE dye and infection was given for 6 h after 24 h of control and infected cell–derived EV treatment (A, upper and lower panel, respectively). Parasite infection was determined by counting the CFSE positive structures inside cells. Scale bar 10 μm. (B) Percentage of infected cells was calculated for both control cell EV and infected cell EV-treated RAW264.7 cells (B, P = 0.0064, unpaired t test, n ≥ 16 number of fields). (C) Parasites were labelled with CFSE dye and infection was given for 6 h after 24 h of pCIneo and pmiR-146a–transfected cell–derived EV treatment. Percentage of infected cells was calculated for both pCIneo and pmiR-146a transfected cell–derived EV-treated cells (C, P = 0.0208, unpaired t test, n ≥ 18 number of fields). (D) Parasites were labelled with CFSE dye and infection was given for 24 h to pCIneo and pmiR-146a–transfected cell. Percentage of infected cells was calculated for both pCIneo and pmiR-146a transfected cells (P = 0.0401, unpaired t test, n ≥ 16 number of fields). (E) RAW264.7 cells were infected with Ld for 24 h and infection was measured in terms of amastin mRNA level by qRT-PCR (E, P = 0.2213, n = 4 independent experiments). (F) Relative level of amastin mRNA was determined by qRT-PCR from control and infected cell–derived EV-treated cells followed by 6 h infection (F, P = 0.0205, n = 4 independent experiments). (G) Relative level of amastin mRNA from pCIneo and pmiR-146a transfected cells after 24 h of infection was determined by qRT-PCR (G, P = 0.0160, n = 4 independent experiments). GAPDH was used as endogenous control. Values for uninfected cell or control EV-treated cell or pCIneo transfected cells were considered as units. (H) RAW264.7 cells were co-transfected with DsRed and pmiR-146a and was infected with Ld for 24 h and infection level was visualized at individual cells (lower panel) and quantified (upper panel). Percent of infection in pmiR-146a/DsRed expressing versus non-transfected Ld-infected cells were measured. Scale bar 10 μm. (n = 38, Number of fields). (I, J, K, L) Latex bead phagocytosis after EV treatment. Red fluorescent bead (Fluorescent Red; Sigma-Aldrich) was diluted in medium and added to EV-treated RAW264.7 cells at 1:10 (cell:bead) ratio for 6 h. Red fluorescent bead was visualized by a confocal microscope. (I, J) Phalloidin 488 was used for staining the cytoskeleton of cells (I). Scale bar 10 μm. Percentage of beads phagocytosed in EV-treated macrophages was plotted and compared between control cell EV-treated and Ld-infected cell EV-treated cells (J, unpaired t test, P = 0.0347, Number of fields, n ≥ 13 number of fields). (K, L) In panel (K) visualization of beads phagocytosed in EV-treated macrophages was monitored in pCIneo transfected control cell EV-treated and pmiR-146a–expressing cell EV-treated cells. The quantification was completed and plotted in panel L (unpaired t test, P = 0.6761, Number of fields, n ≥ 13 number of fields). Scale bar = 10 μm. (M) Cellular levels of the major signalling pathway components in pmiR-146a and pCIneo expression conditions were determined by Western blot. β-Actin was used as loading control. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsignificant, *, ** represent P-value of <0.05, <0.01, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.
Supplementary Materials
Table S1. Information on proteins that are identified in the EVs-isolated from control and infected macrophage cells. The summary of the mass sequencing analysis results are also provided. [LSA-2021-01229_TableS1.xls]
Table S7 Antibodies used for Western Blot.
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