Differential role of gp130-dependent STAT and Ras signalling for haematopoiesis following bone-marrow transplantation

Abstract

Introduction: Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved.

Methods: Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130(ΔMx)), or to selectively disrupt gp130-dependent Ras (gp130(ΔMxRas)) or STAT signalling (gp130(ΔMxSTAT)) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry.

Results: BM derived from gp130 deficient donor mice (gp130(ΔMx)) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130(ΔMxRas) and gp130(ΔMxSTAT) donor BM. BMT of gp130(ΔMxSTAT) cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130(ΔMxRas) BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function.

Conclusion: Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert distinct functions on individual bone-marrow-lineages.

Figure 1. Delayed engraftment of gp130^ΔMx donor BM in the early phase after BM transplantation (BMT).

Numbers of total white blood cells (WBC) [G/l] after BMT: Displayed are total white blood cell counts after BM transplantation at the indicated time points (2, 4, 6 weeks). Less total white blood cells can be detected in gp130^loxP/loxP mice that were transplanted with GFP(+)gp130^ΔMx BM compared to GFP(+)gp130^loxP/loxP donor mice. A) Engraftment of CD45(+)GFP(+) white blood cells (WBC) [G/l] after BMT: Displayed is an example flow cytometry plot 2 weeks after BMT of gp130^loxPloxP BM. Gating on all white blood cells in the FSC/SSC is followed by gating on CD45+ cells. The GFP+ (donor) fraction is shown as a histogram. B) Delayed engraftment of CD45(+)GFP(+) white blood cells (WBC) [G/l] after BMT of gp130 deficient BM: An example flow cytometry plot 2 weeks after BMT of gp130^ΔMx BM is demonstrated. Gating on all white blood cells in the FSC/SSC is followed by gating on CD45+ cells. The GFP+ (donor) fraction is shown as a histogram. Comparison to Fig. 1B shows the decreased GFP+, donor derived, cell fraction. C) Gp130 deficiency in donor mice leads to thrombocytopenia: Depicted are the platelet counts 2, 4 and 6 weeks after BMT. Gp130^loxP/loxP littermates that received GFP(+)gp130^ΔMx BM showed a significant thrombocytopenia 2 weeks after BMT. D) Haemoglobin values [g/dl] after BM transplantation: Haemoglobin values 2, 4 and 6 weeks after BM transplantation are depicted with a significant difference 4 weeks after BMT. [***p<0.001].

Figure 2. Subgroup analysis of different T-cell subsets after BMT.

A)/B) Engraftment of CD4(+)/GFP(+) and CD8(+)/GFP(+) T-cells [G/l]: CD4(+)/GFP(+) (upper plots) and CD8(+)/GFP(+) T-cells (lower plots) were analysed by flow cytometry analysis 2, 4 and 6 weeks after BM transplantation. A lower percentage of CD4(+)/GFP(+) and CD8(+)/GFP(+) T-cells could be detected in gp130^loxPloxP recipients that were transplanted with GFP(+)gp130^ΔMx donor BM. Displayed are example flow cytometry plots for a recipient mouse of wildtype donor BM (A) as well as for a recipient animal of gp130 deficient BM (B) 2 weeks after BMT. C)/D) Engraftment of CD19(+)/GFP(+) B-cells [G/l]: CD19(+)/GFP(+) B-cells derived from GFP(+)gp130^ΔMx donor BM (Fig. 2D) engrafted decelerated compared to GFP(+)gp130^loxPloxP donor BM (Fig. 2C) 2 and 4 weeks after BM transplantation. Shown are example flow cytometry plots 2 weeks after BMT. E)/F) Engraftment of CD11b(+)/GFP(+) cells [G/l]: 2 weeks after BM transplantation the percentage of CD11b(+)/GFP(+) cells was lower in gp130^loxP/loxP recipients transplanted with GFP(+)gp130^ΔMx donor BM compared to controls. Displayed are example flow cytometry plots for 2 weeks transplanted mice having received wildtype (E) or gp130 deficient (F) BM respectively.

Figure 3. Dissection of intracellular gp130 signalling pathways.

A) Number of total white blood cells (WBC) [G/l] after BMT: Displayed are the total white blood cell counts after BM transplantation at the indicated time points (2, 4 and 6 weeks). Less total white blood cells could be detected in gp130^loxP/loxP mice that were transplanted with GFP(+)gp130^ΔMxSTAT BM. Transplantation of GFP(+)gp130^ΔMxRas donor BM did not result in any significant difference concerning the total WBC count. B) Delayed engraftment of CD45(+)/GFP(+) white blood cells (WBC) [G/l] after BMT is STAT-dependent: Displayed are the CD45(+)/GFP(+) (donor derived) cells after BM transplantation at the indicated time points (2, 4 and 6 weeks). Whereas transplantation of GFP(+)gp130^ΔMxRas donor BM into gp130^loxP/loxP animals did not lead to a delayed engraftment of CD45(+)/GFP(+) cells, transplantation of gp130^ΔMxSTAT into gp130^loxP/loxP resulted in a significant decrease of CD45(+)/GFP(+) cells. C) Defective Ras and STAT signalling in donor mice leads to thrombocytopenia: Depicted are the platelet counts 2, 4 and 6 weeks after BMT. Transplantation of GFP(+)gp130^ΔMxRas as well as GFP(+)gp130^ΔMxSTAT donor BM led to a significant thrombocytopenia 2 weeks after BM transplantation. D) STAT-deficiency in donor mice leads to anaemia after BM transplantation: Depicted are the haemoglobin values 2, 4 and 6 weeks after BM transplantation with a significant anaemia in gp130^loxP/loxP recipients transplanted with GFP(+)gp130^ΔMxSTAT donor BM 4 weeks after BMT. [**p<0.01, ***p<0.001].

Figure 4. Subgroup analysis of different T-cell subsets after BMT.

A) Engraftment of CD4(+)/GFP(+) T-cells [G/l]: CD4(+)/GFP(+) T-cells were analysed by flow cytometry analysis 2, 4 and 6 weeks after BM transplantation. A significant lower number of CD4(+)/GFP(+) T-cells could be detected in gp130^loxPloxP recipients that were transplanted with GFP(+)gp130^ΔMxSTAT donor BM. Transplantation of GFP(+)gp130^ΔMxRas donor BM resulted in the same number of CD4(+)/GFP(+) T-cells as transplantation of gp130^loxP/loxP BM into gp130^loxP/loxP littermates. B) Engraftment of CD8(+)/GFP(+) T-cells [G/l]: The absolute number of CD8(+)/GFP(+) T-cells was determined by flow cytometry analysis 2, 4 and 6 weeks after BM transplantation. The number of CD8(+)/GFP(+) T-cells was significantly lower in wildtype mice that were transplanted with GFP(+)gp130^ΔMxSTAT 2 and 4 weeks after BMT. C) Engraftment of CD19(+)/GFP(+) B-cells [G/l]: CD19(+)/GFP(+) B-cells derived from GFP(+)gp130^ΔMxSTAT donor BM engrafted into recipient mice with significant differences at the 2 and 4 week time point. Gp130^loxP/loxP recipients of GFP(+)gp130^ΔMxRas donor BM also displayed a somewhat delayed engraftment 2 weeks after BMT but recovered faster as shown 4 and 6 weeks after BMT. D) Engraftment of CD11b(+)/GFP(+) cells [G/l]: Transplantation of GFP(+)gp130^ΔMxRas donor BM into gp130^loxP/loxP resulted in a significant increase of CD11b(+)/GFP(+) cells at all indicated time points (2, 4, 6 weeks) after BMT. GFP(+)gp130^ΔMxSTAT donor BM led to the same number of CD11b(+)/GFP(+) cells as the transplantation of GFP(+)gp130^ΔMx donor BM. [*p<0.05, **p<0.01].

Figure 5. Survival analysis after BM transplantation.

A) Survival curve after BMT of 1×10⁶ donor cells (6 mice per group): Recipient mice of all different donor genotypes survived BMT of 1×10⁶ donor cells. B) Survival curve after BMT of 2×10⁵ donor cells (8 mice per group): gp130^ΔMxSTAT transplanted mice showed a 75% survival after BMT with 2×10⁵ donor cells. All the other animals survived BMT to 100%. C) Survival curve after BM transplantation using the amount of 5×10⁴ donor cells (6 mice per group): Transplantation of gp130^loxP/loxP donor BM into gp130^loxP/loxP recipient mice led to a survival rate of 100%. 75% of gp130^loxP/loxP recipients of gp130^ΔMxRas donor BM survived the experiment. However, if gp130^loxP/loxP recipients were transplanted with gp130^ΔMx BM, they survived in 33%. Finally, transplantation of gp130^ΔMxSTAT donor BM led to 100% mortality with no (0%) surviving recipient mice. [*p<0.05].