Fig 2: Rad21 mediates differentiation of HSPCs in response to inflammation through NF-κB–dependent signaling. (A) Analysis of DEGs in two RNA-seq experiments: (1) on GFP+CD48−Sca-1+ cells purified from cultured LSK cells, 3 d after isolation and lentiviral infection with an shRNA targeting Rad21 or a control shRNA (luciferase = luci) and (2) on freshly isolated GFP+ LSK cells from mice that were transplanted with lentivirally infected LSK cells (shRNA-Rad21 or shRNA-luci), 4 mo after transplantation. KEGG pathway analysis shows the 14 most down-regulated pathways in Rad21 knockdown cells versus control cells. The analysis did not identify significantly (q < 0.1) up-regulated pathways. Green asterisks highlight NF-κB–related pathways. DEGs in these pathways from the in vivo analysis are depicted in B. (B) The heat map shows DEGs based on GO-terms related to the KEGG pathways highlighted in A (in vivo) in freshly isolated Rad21 knockdown LSK cells (shRad21) versus control (shLuci) LSK cells reisolated 4 mo after LSK cell transplantation. The heat map shows row-scaled and variance-stabilized expression counts for each gene. Red asterisks highlight inflammatory receptor genes. (C) The seven top transcription factor complexes, regulating DEGs in the experiment on culture-activated LSK cells (Fig. S2 A), were identified with the help of the GeneTrail web service. All significant DEGs were sorted according to their fold change and used as input for GeneTrail with default parameters. An adjusted P value cutoff of 0.05 was applied to select the most significant enriched/depleted transcription factor complexes, regulating these DEGs. Seven depleted transcription factor complexes (q < 0.05) were identified in shRad21-LSK cells. (D and E) 5,000 GFP+LSK cells were isolated from the recipient mice of Rad21 knockdown or control LSK cells at 4 mo after transplantation and cultured for 3 d. The percentage of CD11b+ cells in the cultures was determined by FACS. (D) Representative FACS plots showing percentages of CD11b+ cells in the indicated groups. (E) The histogram shows the percentage of CD11b+ cells in the LSK cell cultures in the indicated groups (in total, n = 8 mice per group were analyzed in n = 2 independent experiments). Statistical significance was assessed with two-way ANOVA followed by Tukey’s multiple comparison test on logit-transformed data. (F) LSK cells from 16-mo-old NF-κB1−/− or wild-type mice were infected with viruses carrying shRNAs against Rad21 or luciferase and cultured with or without LPS/Pam3 for 3 d. The graphs depict the increase in differentiation of LSK cells into myeloid cells (CD11b+) in LPS/Pam3-treated cultures relative to PBS-treated cultures of LSK cells from wild-type mice (left) and LSK cells from NF-κB1−/− mice (right) that were infected with the indicated shRNAs. The dots represent individual mice (in total, n = 6 wild-type mice and n = 4 NF-κB1−/− mice were analyzed in n = 2 independent experiments). Statistical significance was assessed with paired t test after log transformation. All data represent mean ± SD; *, P < 0.05; ****, P < 0.0001; ns, not significant.

Fig 3: Aging increases the ground-stage activity of NF-κB signaling in HSPCs. (A) Representative Western blot showing the level of phospho-NF-κB p65 (Ser536) in LSK cells from young (2–3 mo old) and old (24 mo ­old) mice (n = 3 mice per pool per lane for each experiment, n = 2 independent experiments, one of the two experiments is shown; the other experiment shows a similar result). (B and C) Mean fluorescence intensities (MFI) determined by FACS for IL-6R and TLR4 expression on freshly isolated My-biased HSCs, Ly-biased HSCs, and MPPs from young (2–3 mo old) and old mice (22–24 mo old). The box plots represent the interquartile range (25–75%), with the median; whiskers correspond to min and max values. The dots indicate individual mice (in total, n = 5–8 mice per group were analyzed in n = 2 independent experiments). My-biased HSC: CD150hiCD34−LSK; Ly-biased HSC: CD150loCD34−LSK; MPP: CD34+LSK. (D) mRNA expression of Il6 relative to Actb was analyzed in freshly isolated HSCs from young (2 mo old) and old (24 mo old) mice (in total, n = 8 mice per group were analyzed in n = 2 independent experiments). HSC: CD150+CD34−LSK. (E and F) Young (3 mo old) and old (24 mo old) wild-type mice received an i.p. injection of LPS (1.5 mg/kg) and were sacrificed 3 h later. c-Kit+–enriched BM cells were isolated and cultured for 4 h with secretion inhibitor (Brefeldin A). The level of IL-6 in the HSC population was measured by FACS (n = 3–4 mice per group were used in total in n = 2 independent experiments). (E) The histogram depicts the percentages of IL-6–positive HSCs of the indicated age groups. (F) Representative FACS profiles showing the level of IL-6 in indicated groups.(B–E) Statistical significance was assessed by using the Welch’s t test after log transformation (B–D) or with the two-way ANOVA followed by Tukey’s multiple comparison test on logit-transformed data (E). All data represent mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Fig 4: mRNA expression signatures of MPPs upon in vivo LPS stimulation. (A–C) NanoString analysis was performed on MPPs (CD34+LSK) from young (2 mo old) and old (24 mo old) mice, 24 h after injection of LPS (1.5 mg/kg body weight) or control, PBS injection (n = 12 young mice in three pools of 4 mice per pool, and n = 3 old mice as individual samples; the same experiment as depicted in Fig. 6, D–I). The heat maps show the clustering of the indicated groups according to changes in the mRNA expression of inflammatory genes (A), differentiation factors (B), and cohesin genes and cohesin regulator genes (C).

Fig 5: Trauma injury downregulates CD16, TLR2, and TLR4 expression in circulating PMN in patients.(A–D) MFI of CD16, TLR2, TLR4, and CD66b in circulating PMN collected from control patients (n = 6) and trauma patients on day 0 or 1 (n = 9) measured by flow cytometry. A representative flow cytometry analysis image is shown on the right (except for CD66b). Gray represents unstained cells, blue the control, and red the trauma. All data are presented as mean ± SEM. Statistical analysis was performed by using unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01.