Journal: Nature Cell Biology

Article Title: Stromal cell senescence augments haematopoietic cell fitness in clonal haematopoiesis

doi: 10.1038/s41556-026-02025-4

Figure Lengend Snippet: a , Schematic of the experimental design of busulfan-conditioned recipient mice transplanted with Dnmt3a R878H/+ Mx-Cre or control donor haematopoietic cells. Transplant was performed >5 weeks after poly(I:C) treatment of donor mice. b , Frequency of TNFR1 + (left), TNFR2 + (centre) and IL6R + (right) MSCs (CD45 − Ter119 − CD31 − CD51 + PDGFRα + LepR + ) in mice with control or Dnmt3a R878H/+ haematopoiesis. P values determined by unpaired, two-tailed t test ( n = 9 control, n = 10 Dnmt3a R878H/+ biological replicate mice). Non-significant P values ( P > 0.05) not shown. c , Frequency of TNFR1 + (left), TNFR2 + (centre) and IL6R + ( right) ECs (CD45 − Ter119 − CD31 + ) in mice with control or Dnmt3a R878H/+ haematopoiesis. Not significant by unpaired, two-tailed t test ( n = 9 control, n = 10 Dnmt3a R878H/+ biological replicate mice). d , Frequency of pStat3 + (left) and ROS (CellROX) MFI (right) in MSCs in mice with control or Dnmt3a R878H/+ haematopoiesis. P values determined by two-tailed Mann–Whitney test ( n = 5 biological replicates per condition). e , Frequency of pStat3 + (left) and CellROX MFI (right) in ECs in mice with control or Dnmt3a R878H/+ haematopoiesis. Not significant ( P > 0.05) by two-tailed Mann–Whitney test ( n = 5 biological replicates per condition). f , GSEA of ROS, DNA damage response and p53 signatures in MSCs, EC-arteriolar and EC-sinusoidal clusters in mice with Dnmt3a R878H/+ versus control haematopoiesis. Dots shown are FDR < 0.05 ( n = 4 biological replicate mice per condition). g , Schematic of the experimental design for treatment of wild-type MSCs with TNF-α or IL-6 ex vivo. h , Frequency of pStat3 + (left) and CellROX MFI (right) in MSCs after treatment with TNF-α or IL-6. P values were determined by one-way ANOVA with uncorrected Fisher’s least significant difference (LSD). For pStat3: n = 14 vehicle, n = 7 TNF-α, n = 6 IL-6 biological replicates. For CellROX: n = 13 vehicle , n = 8 TNF-α, n = 7 IL-6 biological replicates. i , Frequency of pStat3 + (left) and CellROX MFI (right) in BMECs after treatment with TNF-α or IL-6. Not significant ( P > 0.05) by one-way ANOVA. For pStat3: n = 7 biological replicates per condition. For CellROX: n = 7 vehicle, n = 7 TNF-α, n = 6 IL-6 biological replicates. j , Schematic of the experimental design for treatment of wild-type MSCs with CM from control or Dnmt3a R878H/+ HSPCs with or without the addition of inhibitors targeting TNF-α (etanercept), IL-6 (elsilimomab) or Stat3 (Stattic). k , Representative histogram (top) and MFI (bottom) of SA-β-gal in MSCs treated with CM from control or Dnmt3a R878H/+ HSPCs, with or without the TNF-α inhibitor etanercept (left), IL-6 inhibitor elsilimomab (centre) or the Stat3 inhibitor stattic (right). P values determined by one-way ANOVA with Tukey’s multiple comparisons test. Left (etanercept): n = 3 biological replicates per condition. Centre (elsilimomab): n = 4 biological replicates per condition. Right (Stattic): n = 4 biological replicates per condition. Non-significant P values ( P > 0.05) not shown. l , Proposed model of senescence induction in MSCs involving IL-6 and/or TNF-α produced by Dnmt3a -mutant haematopoietic cells stimulating a Stat3-ROS signalling mechanism in MSCs. Dots represent individual biological replicate mice or human samples. In box plots, the box represents the interquartile range, the median is indicated by the line inside the box, and whiskers extend to the minimum and maximum values. Panel l created in BioRender; Trowbridge, J. (2025) https://BioRender.com/fwgfp4p .

Article Snippet: For inhibitor assays, MSCs were cultured with HSPC CM for five days with 10 μg ml −1 elsilimomab (MedChemExpress), 1 μg ml −1 etanercept (Millipore Sigma) or 5 μM Stattic (MedChemExpress).

Techniques: Control, Two Tailed Test, MANN-WHITNEY, Ex Vivo, Produced, Mutagenesis

Journal: Advanced Science

Article Title: S100A8/A9‐High Macrophages Activate Intestinal Fibroblasts via mCCL6/hCCL15‐CCR1 Axis to Drive Intestinal Fibrosis in Crohn's Disease

doi: 10.1002/advs.76353

Figure Lengend Snippet: S100A8/A9 hi macrophages mediate CCL6 expression through activation of the transcription factor STAT3. (A) Venn diagram integrating multiple bioinformatic databases to identify potential upstream transcription factors regulating Ccl6 expression. (B) Protein‐protein interaction network between the nine transcription factors and S100A9. (C–F) BMDMs were stimulated with recombinant S100A8‐S100A9 protein (1 µg/mL) with or without pretreated with FPS‐ZM1 (1 µ m ), TAK‐242 (1 µ m ) or DMSO vehicle for 1 h. p‐STAT3 and total STAT3 were assessed by Western blot at 2 h (C and D), and CCL6 levels in culture supernatants were measured by ELISA at 24 h (E). The colocalization of S100A8/A9 with TLR4 in BMDMs was assessed by immunofluorescence staining (F); Scale bar, 20 µm. (G,H) Following pretreatment with or without Stattic (5 µ m ) for 1 h, BMDMs were stimulated with recombinant S100A8‐S100A9 protein (1 µg/mL). p‐STAT3 and total STAT3 levels were assessed by Western blot at 2 h (G), and CCL6 in culture supernatants was quantified by ELISA at 24 h (H). (I,J) BMDMs were transfected with siNC or si S100a9 , followed by treatment with Colivelin TFA (50 µg/mL) for 4 h to activate STAT3. Protein levels of S100A9, p‐STAT3/STAT3, and CCL6 were measured by Western blot (I), and p‐STAT3 expression was visualized by immunofluorescence (J; Scale bar: 500 µm). (K) Western blot analysis of p‐STAT3 levels in nuclear and cytoplasmic fractions of BMDMs transfected with siNC or siS100a9 . (L) Schematic representation of putative STAT3 binding sites within the Ccl6 promoter. (M) Dual‐luciferase reporter assays were performed in HEK 293T cells co‐transfected with a control vector (NC) or a Stat3 expression plasmid, together with reporter vectors pGL1‐Control, pGL1‐Ccl6 wild‐type (WT), or pGL1‐Ccl6 mutant (mut). Promoter activity was measured and normalized. N, O) BMDMs were treated with recombinant S100A8‐S100A9 protein (1 µg/mL) for 2 h. STAT3 recruitment to the Ccl6 promoter was analyzed by chromatin immunoprecipitation (ChIP) assay. The enrichment of p‐STAT3 at the promoter region was quantified by RT‐qPCR and expressed as a percentage of the total input (N). Representative agarose gel images confirmed the specificity of the PCR amplification (O). All values are expressed as mean ± SD. ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To investigate the role of the STAT3 pathway in S100A8/A9‐mediated CCL6 production, BMDMs were stimulated with recombinant S100A8‐S100A9 heterodimer (1 μg/mL; HY‐P71076; MCE, USA) for 2 h. To identify the functional receptors mediating S100A8/A9‐induced STAT3 activation, BMDMs were pretreated with the TLR4 inhibitor TAK‐242 (1 μM; HY‐11109; MCE, USA) or the RAGE inhibitor FPS‐ZM1 (1 μM; HY‐19370; MCE, USA) for 1 h. For STAT3 pathway blockade, BMDMs were pretreated with the STAT3 inhibitor Stattic (5 μM; HY‐13818; MCE, USA) for 1 h before stimulation.

Techniques: Expressing, Activation Assay, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Transfection, Binding Assay, Luciferase, Control, Plasmid Preparation, Mutagenesis, Activity Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Amplification