Fig 2: IL-33- or ST2-deficient BMDMs and lung epithelial cells were more proinflammatory. Shown are representative results from two independent experiments with 3 or 7 mice per group. (a) BMDMs were cultured from WT and IL-33-deficient (IL-33-/-) or ST2-deficient (ST2-/-) mice. KC mRNA levels in cells and protein levels in their culture supernatant were measured by qPCR and ELISA respectively. (b) Expression of pro-SPC on isolated primary alveolar epithelial cells was determined by immunofluorescence to assess the purity of the cells; original magnification 400× (top); scale bar, 50 µm. (c) Primary alveolar epithelial cells were isolated and cultured from WT and IL-33-/- or ST2-/- mice. KC mRNA levels in cells and protein levels in their culture supernatant were measured by qPCR and ELISA respectively. (d, e) 16HBE cells were transfected with human IL-33 sgRNA CRISPR/Cas9 All-in-One Lentivector or blank vector plasmids for 72 h. (d) The protein level of IL-33 in transfected 16HBE cells was analysed by Western blotting, (e) and the mRNA level of CXCL1, known as KC in mice, was detected by qPCR (n = 3/group). (f) Expression of p-NF-?B and NF-?B was determined by Western blotting in untreated BMDMs from WT, IL-33-/- and ST2-/- mice. (g) The expression and location of NF-?B were analysed by immunofluorescence in untreated WT, IL-33-/- and ST2-/- primary alveolar epithelial cells. (h, i) Blood and BALF cells were collected after the mice were intratracheally treated with 10 µg LPS for 24 h. (h) Frequencies of CXCR2 expression in circulating neutrophils (i) and mean fluorescence intensity (MFI) of CXCR2 expression in BALF neutrophils were analysed by flow cytometry. Bar graphs and data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. ns, not significant.

Fig 3: CXCL1 and CXCL5 signaling through CXCR2 enhances growth of leukemic stem and progenitor cells(A) Representative flow plots showing CXCR2 expression in purified HSCs from normal (NL) and LSC CML mice without in vitro culture (fresh) or cultured in the presence or absence of CXCL1 (250 ng/mL), measured at the indicated time points.(B and C) Mean fluorescence intensity (MFI) of CXCR2 on CML LSCs (B) and normal (NL) HSCs (C) (n = 4 independent samples/group).(D) FACS-purified CFSEmax LSCs were cultured in the presence or absence of CML 6C3+ cells, CXCL1 (250 ng/mL), CXCL5 (250 ng/mL), CXCL1 and CXCL5, or Veh with or without CXCR2i (10 µM) for 72 h (2,000 cells/condition), and their proliferation and cell numbers were analyzed.(E–H) Absolute number of total CD45+ cells (E), LSCs (F), STHSCs (G), and MPPs (H).(I) Experimental schema of limiting dilution competitive repopulating units (CRU) assay. FACS-purified LSCs cultured alone (CTRL) or with CXCL1 (250 ng/mL) for 72 h were transplanted in limiting doses along with helper BM cells (2.5 × 105 cells/mouse) into recipient mice irradiated at 8 cGy and followed for long-term donor chimerism in the PB for 16 weeks.(J) Table representing the number of recipients that were positive for long-term reconstitution from the limiting dilution CRU assay (n = 8 recipients/cohort per cell dose, n = 4 donors/cohort).(K) Line graph displaying estimates of LSC frequency in the indicated groups, with dashed lines representing 95% confidence intervals. Stem cell frequency and significance were determined using Extreme Limiting Dilution Analysis (ELDA) software.Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: TNF-a signaling contributes to expansion and altered gene expression in 6C3+ stromal progenitors in CML BM(A) Experimental schema. BCR-ABL expression was induced in SCL-tTA-BCR-ABL (CD45.1/45.2+) mice (CML) by tetracycline withdrawal for 8 weeks. BM cells from induced CML mice and aged-matched WT mice (normal) were injected into 6- to 10-week-old WT mice irradiated at 8 Gy (n = 5–6 mice/group). 8 weeks after transplantation, normal and CML mice were treated with Veh or recombinant TNF-a (0.5 µg/mouse; rec-TNF-a) or anti-TNF-a antibody (10 mg/kg, TNF-a inhibitor) once daily intraperitoneally (i.p.) for 2 weeks, after which mice were euthanized and PB and BM were analyzed.(B and C) Absolute number of 6C3+ cells in the BM of mice engrafted with normal cells (B) and CML cells (C).(D and E) Absolute number of THY+ cells in the BM of mice engrafted with normal cells (D) and CML cells (E).(F and G) Relative expression of CXCL1 (F) and CXCL5 mRNA (G) determined by qPCR in normal and CML 6C3+ cells upon the indicated treatment regimens (3 biological replicates per group).(H) Relative expression of CXCR2 determined by qPCR in normal and CML LSK stem/progenitor cells upon the indicated treatment regimens.Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: Altered gene expression in CML compared with normal mesenchymal stem and progenitor cell subsets(A) Experimental schema. RNA-seq analysis was performed on FACS-sorted BM MSC subsets from normal and CML mice (8 weeks after leukemia induction) (4 biological replicates per group) and analyzed for differential gene expression, gene set enrichment analysis (GSEA), and ligand-receptor interactions.(B–E) Volcano plot showing differentially expressed genes in FACS-sorted HSCs and LSCs (B), normal and CML 6C3+ cells (C), normal and CML THY+ cells (D), and normal and CML mSSC cells (E) (fold change = 2, q < 0.05).(F and G) GSEA of differentially expressed genes showed that CML 6C3+ cells exhibited significantly increased expression of multiple gene sets related to inflammatory response (F), including TNF-a-related genes (highlighted in red), and CML THY+ cells exhibited decreased expression of TNF-a-related genes (highlighted in blue) (G), with normalized enrichment score (NES) shown.(H) Heatmap of cytokines and chemokines differentially expressed among normal and CML 6C3+ cells.(I) Heatmap of cytokines and chemokines differentially expressed among CML mSSC, CML THY+ and CML 6C3+ cells.(J–M) Pooled data from RNA-seq showing expression of CXCL1 and CXCL5 in normal and CML 6C3+ cells (J), THY+ cells (K), mSSC cells (L), and CXCR2 expression in HSCs and LSCs (M).(N) Ligand-receptor interaction and fold change of interacting partners between CML LTHSC and CML 6C3 cells compared with normal LTHSC and normal 6C3 cells.n.d., not determined. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.