Therapeutic Targeting of the Inflammasome to Mitigate Murine Sclerodermatous Chronic Graft Versus Host Disease

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the important therapeutic strategies for haematological malignancies. However, developing graft-versus-host disease (GVHD) as a major complication counteracts clinical HSCT benefits. Chronic GVHD (cGVHD) of the skin is often characterized by chronic inflammation and fibrosis. Other key players involved in cGVHD pathophysiology include alloreactive donor B and T-cell involvement. The inflammasome is a multimeric protein complex which can trigger inflammatory responses in response to the pathogen-associated molecular pattern, damage-associated molecular pattern, or homeostasis-associated molecular pattern. The cascade of events results in a potent inflammation characterized by caspase-1 activation, release of interleukins and cell death, which are critical in the pathogenesis of cGVHD. The inhibition of NLRP3 and NLRC4 inflammasome may be beneficial in enhancing cGVHD pathophysiology. In this study, we evaluated the efficiency of kamuvudine-9 (K-9), a derivative of nucleoside reverse transcriptase inhibitors, as an inflammasome inhibitor (NLRP3 and NLRC4) to reduce sclerodermatous skin cGVHD. Methods: Recipient C57BL/6 mice received total body irradiation (850cGy), followed by infusion of bone marrow and splenocytes of either syngeneic (C57BL/6) or allogeneic (LP/J) mice. In another minor mismatch model, recipient BALB/c mice received total body irradiation (800cGy), followed by infusion of either syngeneic (BALB/c) or allogeneic (B10.D2) bone marrow and splenocytes. Recipients were subsequently treated with K-9 or vehicle control (PBS) from starting week 3 after HSCT until end of week 8. Mice were monitored for clinical signs of GVHD, survival, organ pathology of the skin and expression of inflammatory mediators. Results: K-9 treated allogeneic C57BL/6 recipients showed better survival and significantly improved clinical GVHD scores (week 5: p= 0.0011, week 8: p<0.0001) when compared to allogeneic vehicle control-treated mice. Similar outcomes were seen in the B10.D2 into BALB/c HSCT model (week 5: p=0.0029, week 8: p=0.0253). Moreover, both transplant models showed reduced skin pathology (C57BL/6 recipient: p =0.0001, BALB/c recipient: p =0.0018) upon K-9 treatment indicating the therapeutic efficacy of the drug. A significant reduction in dermal thickness and skin fibrosis (both C57BL/6 and BALB/c recipients p<0.0001) as well as mast cell numbers (p<0.0001) were seen after K-9 treatment. Expression of caspase 8 (p=0.0078) and NLRP3 (p=0.0001) were significantly downregulated in skin tissue sections after K-9 treatment as quantified by immunohistochemistry. We next tested the ability of K-9 to alter T-cell responses, using in vitro mixed lymphocyte reactions, a significant decrease in dose-dependent T-cell proliferation was seen. Moreover, the production of tumor necrosis factor by peritoneal macrophages in response to LPS stimulation was reduced by treatment with K-9. Conclusion: Our results demonstrate that specific inhibition of NLRP3 inflammasome with K-9 has a beneficial role in ameliorating cGVHD of the skin. Future work will focus on molecular transcriptional signatures and metabolomic pathways affected by this novel approach to target cGVHD of the skin to expand not only prevention and therapeutic modalities but also pathophysiologic understanding of this disease.