Therapeutic Potential of a Senolytic Approach in a Murine Model of Chronic GVHD

Abstract

Graft-versus-host disease (GVHD) is a life-threatening systemic complication of allogeneic hematopoietic stem cell transplantation (HSCT) characterized by dysregulation of T and B cell activation and function, scleroderma-like features, and multi-organ pathology. The treatment of cGVHD is limited to the management of symptoms and long-term use of immunosuppressive therapy, which underscores the need for developing novel treatment approaches. Notably, there is a striking similarity between cytokines/chemokines responsible for multi-organ damage in cGVHD and pro-inflammatory factors, immune modulators, and growth factors secreted by senescent cells upon the acquisition of senescence-associated secretory phenotype (SASP). In this pilot study, we questioned the involvement of senescent cell-derived factors in the pathogenesis of cGVHD triggered upon allogeneic transplantation in an irradiated host. Using a murine model that recapitulates sclerodermatous cGVHD, we investigated the therapeutic efficacy of a senolytic combination of dasatinib and quercetin (DQ) administered after 10 days of allogeneic transplantation and given every 7 days for 35 days. Treatment with DQ resulted in a significant improvement in several physical and tissue-specific features, such as alopecia and earlobe thickness, associated with cGVHD pathogenesis in allograft recipients. DQ also mitigated cGVHD-associated changes in the peripheral T cell pool and serum levels of SASP-like cytokines, such as IL-4, IL-6 and IL-8Rα. Our results support the involvement of senescent cells in the pathogenesis of cGVHD and provide a rationale for the use of DQ, a clinically approved senolytic approach, as a potential therapeutic strategy.

Keywords: dasatinib; graft vs. host disease; quercetin; senescence.

Figure 1

Effects of the senolytic combination of DQ on murine physical features following clinical onset of cGVHD. (A) Graphical representation of the study design employed. (B) Representative photographs of mice on day 34 post-transplantation. (C) Progression of the mean body weight of mice in the 3 groups over the time course of the study. One-way ANOVA was used to assess statistical significance (* = p < 0.05). (D) Mean disease severity scores (+/− SEM) over the time course of the study post-transplantation. One-way ANOVA was used to assess statistical significance (*** = p < 0.001, **** = p < 0.0001).

Figure 2

DQ treatment ameliorates cGVHD-associated fibrosis in ear and skin tissues. (A) Mean ear thickness was plotted over the time course of the study following grafting. One-way ANOVA was used for statistical significance (* = p < 0.05 and **** = p < 0.0001). (B–D) Representative images of H&E staining (40×) (B), picrosirius red stain (20×) (C), and col1A1 antibody (40×) (D) in ear tissue sections from syngeneic recipients, allogeneic recipients and allogeneic recipients treated with DQ. Allogeneic mice subjected to DQ treatment displayed less fibrosis (attributed to collagen abundance). (E–I) Representative images of H&E staining (40×) (E), picrosirius red stain (20×) (F), anti-col1A1 (40×) (G), MPO (40×) (H), and p16 (40×) (I) in skin tissue sections from mice in the three groups.

Figure 2

DQ treatment ameliorates cGVHD-associated fibrosis in ear and skin tissues. (A) Mean ear thickness was plotted over the time course of the study following grafting. One-way ANOVA was used for statistical significance (* = p < 0.05 and **** = p < 0.0001). (B–D) Representative images of H&E staining (40×) (B), picrosirius red stain (20×) (C), and col1A1 antibody (40×) (D) in ear tissue sections from syngeneic recipients, allogeneic recipients and allogeneic recipients treated with DQ. Allogeneic mice subjected to DQ treatment displayed less fibrosis (attributed to collagen abundance). (E–I) Representative images of H&E staining (40×) (E), picrosirius red stain (20×) (F), anti-col1A1 (40×) (G), MPO (40×) (H), and p16 (40×) (I) in skin tissue sections from mice in the three groups.

Figure 3

DQ treatment alleviates changes in T cell immune profile during cGVHD. (A) Splenocytes were subjected to FACS analysis with anti-B220-PECy7 to label CD45+ B cells amongst total lymphocyte populations. One-way ANOVA was used for statistical significance (* = p < 0.05, ** = p < 0.01). (B) Frequency of naïve and memory CD4 cells amongst total CD4 population was determined by CD62L and CD44 labeling. Two-way ANOVA was used for statistical significance (* = p < 0.05, ** = p < 0.01, **** = p < 0.0001). (C) Frequency of naïve and memory CD8 cells amongst total CD8 population was determined by CD62L and CD44 labeling. Two-way ANOVA was used for statistical significance (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

Figure 4

DQ treatment modulates cytokine secretion and protein expression associated with GVHD in allogeneic recipients. (A) 15-plex ELISA profiling of key inflammatory cytokines in the three groups of animals. IL-4 and IL-6 levels in the three groups were plotted and two-way ANOVA was used for statistical significance (*** = p < 0.001, **** = p < 0.0001). (B) IL-8Rα secretion in syngeneic and allograft recipients was measured using ELISA. One-way ANOVA was used for statistical significance (* = p < 0.05).

Figure 5

DQ targets senescent cells in liver. (A,B) mRNA expression levels of IL-8 and IL-6 produced by liver, colon, and lung cells were determined by qPCR. Two-way ANOVA was used for statistical significance (* = p < 0.05, ** = p < 0.01, **** = p < 0.0001). (C–E) Representative images of H&E stain (40×) (C), p16 (20×) (D), and CD45 (40×) (E) in liver tissue sections from mice in the syngeneic, allogeneic, and allogeneic + DQ groups.

Figure 6

Senolytic combination of DQ alleviates organ manifestations of cGVHD, particularly skin fibrosis, accumulation of senescent cells in the liver, and alloreactive T cell repertoire. Summary model depicting the proposed mechanism of the physical and molecular pathophenotypes mitigated by targeting the senescence-associated secretory phenotype-driven cytokine storm in cGVHD. (?) denotes the probable sites of action; however, the exact mechanism of senescence inhibitory therapeutic activity of DQ remains unclear.