JAK1/2 inhibitor ruxolitinib promotes the expansion and suppressive action of polymorphonuclear myeloid-derived suppressor cells via the JAK/STAT and ROS-MAPK/NF-κB signalling pathways in acute graft-versus-host disease

Abstract

Objectives: Ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor, demonstrates efficacy for treating steroid-resistant acute graft-versus-host disease (SR-aGVHD) following allogeneic stem cell transplantation (allo-HSCT). Myeloid-derived suppressor cells (MDSCs) have a protective effect on aGVHD via suppressing T cell function. However, the precise features and mechanism of JAK inhibitor-mediated immune modulation on MDSCs subsets remain poorly understood.

Methods: A total of 74 SR-aGVHD patients treated with allo-HSCT and ruxolitinib were enrolled in the present study. The alterations of MDSC and regulatory T cell (Treg) populations were monitored during ruxolitinib treatment in responders and nonresponders. A mouse model of aGVHD was used to evaluate the immunosuppressive activity of MDSCs and related signalling pathways in response to ruxolitinib administration in vivo and in vitro.

Results: Patients with SR-aGVHD who received ruxolitinib treatment achieved satisfactory outcomes. Elevation proportions of MDSCs before treatment, especially polymorphonuclear-MDSCs (PMN-MDSCs) were better to reflect the response to ruxolitinib than those in Tregs. In the mouse model of aGVHD, the administration of ruxolitinib resulted in the expansion and functional enhancement of PMN-MDSCs and the effects could be partially reversed by an anti-Gr-1 antibody in vivo. Ruxolitinib treatment significantly elevated the suppressive function of PMN-MDSCs through reactive oxygen species (ROS) production by Nox2 upregulation as well as bypassing the activated MAPK/NF-κB signalling pathway. Additionally, ex vivo experiments demonstrated that ruxolitinib prevented the differentiation of mature myeloid cells and promoted the accumulation of MDSCs by inhibiting STAT5.

Conclusions: Ruxolitinib enhances PMN-MDSCs functions through JAK/STAT and ROS-MAPK/NF-κB signalling pathways. Monitoring frequencies and functions of MDSCs can help evaluate treatment responses to ruxolitinib.

Keywords: JAK/STAT pathway; ROS; acute graft‐versus‐host disease; myeloid‐derived suppressor cells.

Figure 1

Ruxolitinib treatment improved the overall response of patients with SR‐aGVHD after allo‐HSCT along with alterations of myeloid‐derived suppressor cells (MDSCs). (a) The overall response rate (ORR) at day 28 and day 56 in patients with SR‐aGVHD who received ruxolitinib treatment after allo‐HSCT (n = 74). (b) The overall survival of patients with ruxolitinib for aGVHD according to the response of ruxolitinib (Responders who obtained PR or above, n = 51; Nonresponders who failed treatment, n = 23). (c) The percentages of MDSCs (CD11b⁺HLA‐DR⁻), PMN‐MDSCs (CD11b⁺HLA‐DR⁻CD14⁻CD15⁺), M‐MDSCs (CD11b⁺HLA‐DR⁻CD14⁺CD15⁻), Tregs (CD4⁺CD25⁺Foxp3⁺) from PB samples in responders (n = 7) and nonreponders (n = 6) before and within 7 days, 14 days after starting ruxolitinib. (d, e) Correlation analysis of the MDSCs and PMN‐MDSCs proportions before ruxolitinib administration compared with response to ruxolitinib treatment. Each dot represents an independent core. The response to ruxolitinib included response (CR + PR) and nonresponse (0 = nonresponse, 1 = response, respectively). r and P‐values were calculated using Spearman's correlation test. *P < 0.05, **P < 0.01.

Figure 2

Ruxolitinib treatment reduced aGVHD severity and promoted myeloid‐derived suppressor cells (MDSCs) expansion. Lethally irradiated BALB/c mice were transplanted with 1 × 10⁷ BM cells with or without 3 × 10⁷ splenic cells from C57BL/6 mice. Recipient mice received vehicle or ruxolitinib (30 mg/kg) by oral gavage twice a day after transplantation. (a) Schematic representation of the experimental procedure. The overall survival (b), weight variation (c) and clinical GVHD score (d) were shown in each group (BM‐only group, n = 6; Vehicle group, n = 12; Ruxolitinib group, n = 12). (e) Representative haematoxylin & eosin‐stained sections of the liver, intestine and colon from vehicle and ruxolitinib‐treated mice at day 7 after transplantation (scale bar, 100 μm). The arrows were used to indicate the representative areas. (f–i) The percentages and absolute numbers of donor‐derived splenic MDSCs (CD11b⁺Gr‐1⁺), PMN‐MDSCs (CD11b⁺Ly6G⁺Ly6C^lo) and M‐MDSC (CD11b⁺Ly6G⁻Ly6C^hi) were measured with flow cytometry in vehicle treatment and ruxolitinib treatment groups at day 7 after transplantation (n = 4 or 5 per group). (j) The morphology of splenic‐derived PMN‐MDSCs isolated from vehicle‐ and ruxolitinib‐treated mice at day 7 after transplantation, which were visualised by Wright–Giemsa staining under light microscope (scale bar, 10 μm). *P < 0.05, **P <0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

Ruxolitinib treatment enhanced immunosuppressive function of MDSCs, especially PMN‐MDSCs in aGVHD mice. Depletion of MDSCs exacerbated aGVHD lethality in ruxolitinib‐treated mice. (a–c) CFSE‐labelled CD3+ T cells (1 × 10⁵ per well) from wild‐type C57BL/6 spleen were stimulated with CD3/28 beads and cocultured with different ratios of purified splenic MDSCs, PMN‐MDSCs and M‐MDSCs isolated from vehicle‐ and ruxolitinib‐treated mice at day 7 after transplantation for 72 h. Proliferation of CFSE‐labelled CD3+ T cells was measured with flow cytometry (Vehicle group, n = 3; Ruxolitinib group, n = 3). (d) The helper T cell‐related cytokines were detected in supernatants harvest from the above coculture system (n = 3). (e,f) aGVHD mouse models were built as described previously. Two hundred microgram anti‐Gr‐1 antibody was injected intraperitoneally into recipient mice with ruxolitinib treatment to deplete MDSCs as Gr‐1 depletion group from day 5 to day 29 every other day after transplantation. The overall survival and weight ratio were exhibited in each group (Vehicle group, n = 6; Ruxolitinib group, n = 6; Gr‐1 depletion group, n = 5). Data are expressed as mean ± standard error (SE) and from three independent experiments. **P < 0.01, ***P < 0.001.

Figure 4

Transcriptional signatures and related protein levels of splenic PMN‐MDSCs in vehicle‐ and ruxolitinib‐treated mice at day 7 after transplantation. (a) Gene pathways that were differentially expressed in PMN‐MDSCs from two groups according to Gene Set Enrichment analysis. Gene sets were considered statistically significant at an FDR P‐value < 0.05 (n = 2 per group). (b) Dot graph shows the alterations of enriched GO pathways between two groups. The expression profile of PMN‐MDSCs function‐related genes between two groups according to (c) RNA sequencing and (d) real‐time PCR (n = 2 or 3 per group). (e) Flow cytometric detection of ROS in PMN‐MDSCs of vehicle‐ and ruxolitinib‐treated hosts on day 7 post‐transplantation. Representative DCFDA staining flow cytometry data gated on CD11b⁺Ly6G⁺Ly6C^lo cells are shown. Geometric mean fluorescence intensity (MFI) values are plotted (n = 3). (f, g) The expression of pSTAT3 and pSTAT5 in PMN‐MDSCs of vehicle‐ and ruxolitinib‐treated hosts on day 7 post‐transplantation by phosflow techniques (n = 3). (h) The phosphorylation levels of p65, ERK, p38 and Akt were quantified by western blot assay in cell lysates of PMN‐MDSCs from vehicle‐ and ruxolitinib‐treated mice on day 7 post‐transplantation. β‐Actin was used as an internal control. Data are expressed as mean ± standard error (SE). **P < 0.01, ***P < 0.001, ****P < 0.0001. These results are representative of three independent experiments.

Figure 5

Ruxolitinib‐pretreated PMN‐MDSCs displayed remarkable immunosuppressive function by upregulation of Nox2 to regulate ROS generation via bypass activating NF‐κB/MAPK‐p38 pathways in vitro. MDSCs were generated in vitro from BM cells of C57BL/6 mice in the presence of 40 ng mL⁻¹ GM‐CSF and IL‐6. After 4 days, cells were stained for CD11b and Gr‐1 expression or the distribution of MDSC subsets was gated on CD11b⁺ cells by the expression of Ly6C and Ly6G. (a) Data show one representative flow cytometric analysis and morphology of MDSCs subsets. (b) In vitro purified PMN‐MDSCs were pretreated with or without ruxolitinib (0.1 μm, 1 μm, 10 μm) for 2 h and then incubated with LPS (1 μg mL⁻¹) in the presence or absence of ROS inhibitor NAC (1 mm) for another (h). The production of ROS was monitored by DCFDA flow cytometry in each group (n = 3). (c–e) CFSE‐labelled CD3⁺ T cells (1 × 10⁵ per well) were stimulated by CD3/28 beads, then in vitro induced MDSCs, PMN‐MDSCs and M‐MDSCs were added at different ratios with or without ruxolitinib pretreated cocultured for 72 h. Proliferation of CFSE‐labelled CD3+ T cells was measured with flow cytometry. In vitro purified PMN‐MDSCs were pretreated with or without ruxolitinib (0.1 μm, 1 μm,10 μm) for 2 h and then stimulated with LPS (1 μg mL⁻¹) (n = 3). (f) The immunosuppressive molecules of PMN‐MDSCs were detected by real‐time PCR (n = 3). The expression of pSTAT3, p‐p65, p‐p38, p‐ERK and p‐Akt were examined by phosflow analysis (g) and western blot assay (h). Data are expressed as mean ± standard error (SE). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. These results are representative of three independent experiments.

Figure 6

Ruxolitinib decreased the differentiation of MDSCs to mature cells via STAT5 inhibition in vitro. BM‐derived MDSCs in vitro were cultured with GM‐CSF with or without different concentrations of ruxolitinib (0.1 μm, 1 μm, 10 μm) for 5 days. (a) Representative plots of macrophages (CD11b⁺F4/80⁺) and dendritic cells (CD11b⁺CD11c⁺) were shown. The percentages of differentiated mature cells (b) and the expression of costimulatory molecules CD80⁺ gated on CD11b⁺ cells (c) were indicated (n = 3). (d) The phosphorylation of STAT5 was examined by phosflow techniques (n = 3). (e) The levels of transcriptional factors related to MDSCs differentiation were monitored by real‐time PCR (n = 3). Data are expressed as mean ± standard error (SE). ***P < 0.001, ****P < 0.0001. These results are representative of three independent experiments.