Use of R-spondin1, An Intestinotrophic Mitogen, in the Treatment of Murine Graft-Versus-Host Disease

Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for a variety of hematologic malignancies. However, overall success of HCT is limited due to the immunologic recognition and destruction of host tissues resulting in graft-versus-host-disease (GvHD). In GvHD, donor derived immune cells recognize and destroy recipient epithelial tissues; predominantly liver, gastrointestinal (GI) mucosa, and skin. It has been reported that GI toxicity and crypt loss is a strong predictor of GvHD morbidity and mortality. Therefore, GI protective strategies may diminish GvHD severity. R-spondin1 (Rspo1) is an epithelial mitogen that stimulates mucosal growth in both small and large intestines. Therapeutic administration of Rspo1 has been shown to reduce the loss of body weight, diarrhea and rectal bleeding in experimental mouse colitis models. Histological evaluation revealed that Rspo1 improved mucosal integrity by stimulating crypt cell growth and mucosal regeneration in colitis-induced mice. Because the symptoms and pathogenesis of GI-GvHD are similar to colitis models, we chose to examine the impact of Rspo1 on mice with GvHD. Conventional CD4+/CD8+ T cells (Tconv) isolated from FVB (H-2q) animals were transplanted along with T cell-depleted bone marrow (TCD-BM) into lethally irradiated BALB/c (H-2d) mice. Mice received either 100μg Rspo1 protein in 100μL PBS or 100μL PBS alone IV from day 1 to day 6 post-transplantation. Surprisingly, mice receiving Rspo1 had significantly increased rates of mortality. Mice receiving Rspo1 experienced 100% mortality by day 6 post-HCT. In contrast, mice receiving only PBS demonstrated 0% mortality at 20 days post-HCT. Mortality was not a result of Rspo1 toxicity, as no morbidity or mortality was observed in the absence of allogenic Tconv in mice transplanted with either syngenic HCT or allogeneic TCD-BM alone. In order to assess whether the higher mortality experienced by Rspo1 treated mice was due to an explosive proliferation of Tconv, the transplant experiment was repeated with donor Tconv isolated from luciferase transgenic (luc+) H-2q mice. Groups receiving Rspo1 versus PBS did not show significantly different bioluminescence imaging signal intensity, suggesting that rapid proliferation of donor Tconv was not the basis for the observed differences in mortality. The overall mouse mortality in this experiment was consistent with the previous experiment. In an attempt to uncover the mechanism, we found no differences in the Perforin-Granzyme B or Fas/Fas ligand expression between control and treatment groups. Cytokine milieu (IFNγ, TNFα, IL-17, and IL-2) assessment in transplanted mice did not show significant differences in cytokine profiles of control versus Rspo1 treatment. Mouse autopsy demonstrated that transplanted mice receiving Rspo1 had marked crypt hyperplasia, apoptotic cells, and increased mitoses when compared PBS recipients. All other organ systems were considered normal in both groups by a veterinary pathologist. Blood cultures of both groups were negative for bacteria. CBC, liver enzymes, and electrolyte panels were normal in both groups. In order to assess lethal irradiation as a contributing factor to the increased mortality of Rspo1 treated mice, we repeated the above transplant experiment including non-irradiated immunodeficient Rag2−/−, γ-chain−/− H-2d recipients in addition to the lethally irradiated H-2 d recipients. Our mortality findings in the lethally irradiated group were consistent with the above mentioned transplant experiments showing rapid mortality rates in recipient mice receiving Rspo1. However, non-irradiated Rag2−/− γ-chain−/− mice that received Rspo1 had almost identical mortality rates to those receiving PBS; all mice surviving over 20 days post HCT. This finding underlines the critical importance of lethal irradiation as a contributing factor in Rspo1 treatment mortality. These findings suggest that the beneficial effects of Rspo1 in the colitis model may not be reproducible in GvHD. Furthermore, our studies indicate that the lethal effects of Rspo1 may be due to a myriad of factors including allogenic mismatch and lethal irradiation.