EMBO/SourceData huggingface dataset:
Figure 4(A) Lung ECs were isolated from EC- miR-483 transgenic rats (Tg) and their wild-type littermates (WT), n = 3 in each group. MiR-483-3p/-5p levels were measured by qPCR. Data information: Values are expressed as mean ± SEM.(B) Serum from WT and Tg rats (n = 6) were collected. MiR-483-3p and -5p associated with CD144 were enriched by immunoprecipitation with anti-CD144. Levels of miR-483-3p/-5p were measured by qPCR. Data information: Values are expressed as mean ± SEM. Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. Proliferation of ECs were measured by flow cytometry Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsLung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. migration of ECs were measured by wound healing assay Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsTg and WT rats were injected with saline or MCT. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA and protein in lung tissues were measured by qPCR and Western blot, respectively (E). Data information: Values are expressed as mean ± SEM.Tg and WT rats were injected with saline or MCT. Levels of miR-483-3p/-5p in serum and lung tissues were measured by qPCR (F). Data information: Values are expressed as mean ± SEM.Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated miRNAs (G) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM. Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated mRNAs (H) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM.
Figure 5EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of mPAP for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).Vascularization evaluated by H&E staining of pulmonary arteries (C). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 20 µm (C)Vascular integrity evaluated by intravenous injection of Evans blue dye (D). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 0.5 cm (D).EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of mPAP (E) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of RV hypertrophy [RV/(LV+S)] (F) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).
Figure 6qPCR analysis of miR-483-3p/-5p in serum from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsqPCR analysis of miR-483-3p/-5p in lung tissue from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups(D) Protein and mRNA levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 measured by Western blot and qPCR, respectively (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsAnalysis of mPAP (E) for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsAnalysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groupsAnalysis of systemic blood pressure (SBP) (G) for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups(H) RV hypertrophy shown by the H&E staining and RV/(LV+S) quantification. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Scale bar: 0.5 cm (H)(I) Vascularization revealed by H&E staining (n = 7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Scale bar: 20 µm (I).(J) Survival rate of 3 groups (n = 15 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups
Figure 1A-C 10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. The color scales on the right indicate the radiance, i.e. the sum of the photons per second from each pixel inside the ROI/number of pixels (photons/sec/cm2/sr).10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. D The frequency of involvement of the indicated tissues is shown for one cohort of mice and is representative of two independently analyzed cohorts per cell line.E Gating strategy for the identification of U-2932 cells residing in spleen and bone marrow, among all leukocytes in these organs. Cells were stained for human and mouse CD45. Very few hCD45+ U-2932 cells were detectable in the spleen, whereas their frequencies were much higher in the bone marrow (BM). In BM, >90% of U-2932 cells were positive for ZsGreen. The green curve shows U-2932 cells recovered from BM, overlayed with unlabelled U-2932 cells grown in vitro (grey curve).F, G Frequencies of human lymphoma cells, as judged by hCD45 expression, among all (human and mouse, h+m) CD45+ leukocytes in the spleen (S), femoral bone marrow (BM) and jaw bone marrow (JB) at the study endpoint of the mice shown in D (four weeks post injection for U-2932, five weeks p.i. for RIVA and six weeks p.i. for RC-K8).
Figure 2MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution efficiency (frequency of human among all human and mouse CD45+ leukocytes) is shown in AMISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. the average human leukocyte composition in the bone marrow of the mice is shown in B.MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. Tumor volumes over time are presented in CMISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. tumor weights and volumes at the study endpoint in D, where each data point represents one tumor.MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution level directly correlates with the tumor weight at the endpoint (E).MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the spleen (F) and bone marrow (G) by staining for hCD45 (as well as hCD19 and hCD20 for U-2932 and RC-K8, respectively)MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified by ZsGreen expression and in the case of RC-K8 cells, also over time by IVIS (H).MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The frequency of tissue involvement was assessed by ex vivo IVIS and macroscopic evaluation (I),MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. the occurrence of clinical symptoms (weight loss, hind leg paralysis) was recorded for each mouse (J).
Figure 3MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint (A).MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint (A). The lymphoma burden at the study endpoint (six weeks p.i. for RC-K8 and four weeks p.i. for U-2932) was quantified in the spleen (B) and bone marrow (C) by flow cytometric staining for hCD45 and by ZsGreen expression.MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint The involvement of the indicated tissues as assessed by ex vivo IVIS is shown in D (solid bars, MISTRG; hatched bars, MISTRG6),MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint mice that have developed clinical symptoms (paralysis of hind legs) are plotted in E.F Surface expression of the IL-6 receptor α-chain (CD126) as assessed by flow cytometry of U-2932 and RC-K8 cells. Data is representative of two stainings.
Figure 41x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. The spleen weights (B, representative pictures in A) are shown1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. with representative H&E-stained spleen (MISTRG6) and lymph node (patient) sections in C1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. the lymphoma burden at the study endpoint eight weeks p.i., as quantified in the spleen and bone marrow in D Representative flow cytometry plots are shown in D for bone marrow and spleen (MISTRG, MISTRG6).x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. the lymphoma burden at the study endpoint eight weeks p.i., as quantified in the spleen and bone marrowMISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPC, and intravenously injected with 1x106 primary DLBCL cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the bone marrow of reconstituted and un-reconstituted animals by flow cytometric staining for the indicated surface markers.Primary lymphoma cells harvested at the study endpoint express the IL-6R α chain (CD126, as assessed by flow cytometry, G) and are positive for phosphorylated STAT3 (G).Primary lymphoma cells harvested and the signaling chain gp130 (as assessed by immunohistochemistry, H)
Figure 5The expression of the IL-6Rα (CD126) and signaling chains (gp130) was assessed by flow cytometry (A) on a panel of DLBCL cell lines. ABC- and GCB-DLBCL cell lines are color-coded in red and green; RC-K8 are depicted in grey, as their subtype is a matter of debateThe expression of the IL-6Rα (CD126) and signaling chains (gp130) was assessed by SYBR or Taqman qRT-PCR (B, C) respectively, on a panel of DLBCL cell lines. ABC- and GCB-DLBCL cell lines are color-coded in red and green; RC-K8 are depicted in grey, as their subtype is a matter of debateD and E. The expression of gp130 in 114 tumor biopsies of DLBCL patients was assessed by immunohistochemistry, and correlated with the ABC/non-GCB vs. GCB subtype, as assessed via Hans classification. Representative micrographs are shown in D, alongside the quantification of gp130+ cases among all samples, and among the two subtypes, in E. Scale bar, 100 μm.
Figure 6A IL-6 secretion of the indicated cell lines (ABC-DLBCL in red, GCB-DLBCL in green) and primary patient material, as quantified by ELISA and normalized to cell number (shown per 1000 cells). n.d., not detectable.STAT3 phosphorylation as assessed by Western blotting (B, D) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, with and without 1h of exposure to 50ng/ml hIL-6 (B,D). STAT3 expression levels are shown as Western blot control.STAT3 phosphorylation as assessed by flow cytometry (C) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, Histograms in C represent STAT3 phosphorylation in the absence of exogenously added IL-6.E STAT3 phosphorylation as assessed by Western blotting of CD126-proficient (WT) or -deficient (ko) RC-K8 cells, with and without 1h of exposure to 50ng/ml hIL-6.F Kinetics of STAT3 phosphorylation as assessed by Western blotting, of the IL-6-expressing SOCS1wt and SOCS1mut cell lines OCI-Ly3 and SU-DHL-2, under 1, 6 and 24h exposure to an IL-6-neutralizing antibody (α-IL-6, anti-IL-6 neutralizing antibody).STAT3 phosphorylation and gp130 expression of two representative cases (G) assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression; the p-value was calculated by chi-square test. Scale bar, 100 μm.STAT3 phosphorylation and gp130 expression of two representative cases (G) and summarized for the entire cohort of 114 patients (H), as assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression
Figure 7Disease-specific mortality (A) of cohorts of 9-12 mice each (n indicated in figure) of the indicated genotypes.lymphoma burden as determined by lymph node weight (B), of cohorts of 9-12 mice each (n indicated in figure) of the indicated genotypes.Mice of the indicated genotypes were immunized once with sheep red blood cells and assessed 10 days later by flow cytometry of splenocytes for their frequencies of GC B-cells (C), centroblasts (D) and centrocytes (E).Spleen sections were further stained for Ki67 to reveal GCs (F), which were quantified both in number (G) and area (H). Arrows in F point to GCs; scale bar, 1000 μm.
Figure 8Frequency of STAT3 phosphorylation (A), of DLBCL patients stratified according to subtype and disease stage Phospho-STAT3 positivity was defined as expression in ≥17% of tumor cells and more common found in ABC-DLBCL tumors, and in more advanced (stage 3 and 4, relative to stage 1 and 2) disease (A).overall as well as disease-specific survival probability (B) of DLBCL patients pSTAT3 positivity was a strong negative prognostic factor in the entire cohort (B)overall mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall- as well as disease-specific mortality in both ABC- and GCB-DLBCL subtypesdisease-specific mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall- as well as disease-specific mortality in both ABC- and GCB-DLBCL subtypesEffect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, on (E and F) the spleen weights of MISTRG6 mice transplanted with 1x106 primary DLBCL cells (expanded by one serial passage through MISTRG6 mice),Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, Representative microscopy images in F show STAT3 phosphorylation in spleen-colonizing primary cells exposed to either tocilizumab or isotype control antibody (in brown, counterstained in blue with hematoxylin), and an H&E-stained section from the same mouse. TCZ, tocilizumab. Scale bars, 100 μm.Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the bone marrow lymphoma burden of OCI-Ly3-transplanted MISTRG mice (G) assessed at around four weeks post tumor cell injection.Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the splenic lymphoma burden of RC-K8-transplanted MISTRG6 mice (H), all assessed at around four weeks post tumor cell injection.
Figure 1Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. ISG response measured by ISG15 and RSAD2. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. The column labelled c shows control samples collected directly after purification. Quantification of the presence of SeV or IAV. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Figure 2Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (A) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1 (Donors marked in orange and blue were not quantifiable in all test conditions). (B) ISG response measured by ISG15, RSAD2, and IFIT1.Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (C) Quantification of the presence of SARS-CoV-2.
Figure 3Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (B) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. (C) ISG response measured by ISG15, RSAD2, and IFIT1 (Donors marked in green and red were not quantifiable in all test conditions).Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (D) Quantification of the presence of SARS-CoV-2.Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (E) Western blots were performed using samples from two donors. (A-D) Each colour corresponds to a donor; 5 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Figure 4Alveolar macrophages from different donors were purified from BAL and treated with SARS-CoV-2 for either 2 or 24 hours. (A) The amount of SARS-CoV-2 nucleocapsid phosphoprotein was quantified using LC-MS/MS. (B) After subtraction of the background (unchallenged AMs), the ratio between the amount of SARS-CoV-2 nucleocapsid phosphoprotein at 24 and 2 hours was calculated. Each colour corresponds to a donor; 3 different donors were analyzed. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05.
Figure 5(A) Total RNA was harvested from AMs and primary human airway epithelial cells (HAE) and subjected to qPCR for quantification of the ACE2 receptor. Each colour corresponds to a donor. The dashed line represents the detection limit.(B) The presence of nucleoprotein mRNA of SARS-CoV-2 in AMs and primary HAE was also quantified after SARS-CoV-2 treatment. Each colour corresponds to a donor. An unpaired t-test test was used for statistical analysis: ****, P≤0.0001.(C) Primer efficiency was tested using serial dilutions of positive samples using qPCR. The Ct values were plotted on a logarithmic scale along with the corresponding dilutions before performing a linear regression in order to calculate the slope of the trend line through the data points. This analysis revealed an amplification factor of 1.92, 1.91, and 1.85 for the SARS-CoV-2, IAV, and SeV primer set, respectively. The different colours correspond to different primer sets.
Figure 2(A) Proportion change of Chinese SL isolates from different phylogenetic clades. The Chinese isolates were classified into two groups based on their sampling time (1993-2010 vs 2011-2020). The percentage of isolates from each clade in each period was compared.