Preemptive HMG-CoA Reductase Inhibition Significantly Attenuates a Murine Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a serious and increasingly common complication of allogeneic stem cell transplantation. Treatment of cGVHD remains a large challenge. Even though cGVHD differs among patients, the clinical complications of cGVHD often include fibrosis and scleroderma-like changes. A murine sclerodermatous graft-versus-host disease (Scl GVHD) model sharing critical characteristics with human cGVHD is characterized by skin thickening and lung fibrosis. Statins, 3-hydroxy-3-methyl-CoA (HMG-CoA) reductase inhibitors which are a class of cholesterol lowering drugs decreasing mortality from cardiovascular disease and stroke, have strong immunomodulatory effects on antigen-presenting cells and T cells interfering with synthesis of L-mevalonate and its downstream isoprenoid metabolites. It has been demonstrated that some statins inhibit the production of proinflammatory cytokines and improved chronic inflammatory disorders. We used B10.D2 → BALB/c model of cGVHD, which differ at minor histocompatibility loci, to address the therapeutic effect of statins on the development of cGVHD. Lethally irradiated (700 cGy) BALB/c mice were transplanted with either B10.D2 (allogeneic) or BALB/c (syngeneic) bone marrow (1.5 × 10⁶) and spleen cells (3 × 10⁶) to generate Scl GVHD. We have noted skin thickening as early as day 14, however 28 days was usually required to observe significant skin thickening in allogeneic recipients with Scl GVHD. The skin thickenings were still lingering on but weaken as late as 56 days post-alloSCT, whereas the loss of normal lacy alveolar pattern of lungs remained constant up to that time. Syngeneic recipients did not show any significant changes in each organ. In vivo treatment of pravastatin (30 mg/kg/day, intraperitoneally) for 10 days early after transplant led to greater suppression of the incidence and severity of clinical skin cGVHD compared with allogeneic controls injected with diluent. The occurrence of clinical cutaneous GVHD was significantly slower in onset in the recipients of pravastatin treatment than it in the control animals (36 days vs. 25 days, respectively, P<0.05) Blinded pathologic scoring of skin disease on day 28 confirmed the clinical result. Skin from pravastatin recipients had an average pathology score of 2.5 ± 0.3 compared with 6.9 ± 0.7 for the allogeneic controls (P<0.01). To qualify the cells infiltrating skin or lungs in early Scl GVHD, we examined the number of CD11b⁺ or CD3⁺ cells isolated from each organ of the animals with Scl GVHD with or without pravastatin treatment on day 14, 28 and 56 post-alloSCT. Compared with the allogeneic controls with diluent treatment, the injection of pravastatin significantly reduced the number of cells infiltrating each organ on day 14; for bronchoalveolar lavage fluid 3.2 ± 0.3 ×10⁴ vs. 2.2 ± 0.3 ×10⁴ (P<0.05, CD11b⁺) and 1.3 ± 0.1 ×10⁴ vs. 0.33 ± 0.1 ×10⁴ (P<0.05, CD3⁺), for skin 56 ± 4.3 ×10⁴ vs. 10 ± 2.3 ×10⁴ (P<0.01, CD11b⁺) and 12 ± 3.1 ×10⁴ vs. 3 ± 1.8 ×10⁴ (P<0.01, CD3⁺). On days 28 and 56, the infiltrating cells were also significantly reduced in the animals treated with pravastatin (data not shown). To test whether statins might produce an effect on attracting these monocytes to skin or lungs by influencing the chemokine expression, we performed semiquantitative RT-PCR analyses and ELISA on day 14 to examine the expression of MCP-1 and RANTES in skin and lungs from Scl GVHD animals treated by either pravastatin or the diluent. MCP-1 and RANTES mRNA levels as well as protein concentrations from each organ were significantly reduced in recipients treated with pravastatins compared to the allogeneic controls. In conclusion, preemptive HMG CoA reductase inhibition prevented murine Scl GVHD by effectively blocking the influx of monocytes into target organs and by down-regulating the expression of MCP-1 and RANTES, thereby reducing new collagen synthesis. The Scl GVHD model is valuable for testing the effect of statins on early fibrosing diseases, and chemokines may be the potential targets in cGVHD protection effect of statins.