TNFAIP8 (TIPE) Deficiency Exacerbates Acute Graft Versus-Host Disease in Murine Model

Background: Acute graft-versus-host disease (GVHD) occurs after allogeneic hematopoietic cell transplant (alloHCT) and presents a donor immune cell response against host tissues associated with significant morbidity and mortality. Up to 60% of alloHCT recipients will develop acute GVHD. Damage to the gastrointestinal tract from the conditioning is a key step in its pathogenesis by allowing for systemic translocation of lipopolysaccharide (LPS) and other danger signals, hereby promoting host APC activation. Tumor necrosis factor (TNF)--induced protein 8 (TNFAIP8) family is a newly identified group of proteins consisting of TNFAIP8 (TIPE), TIPE1 (TNFAIP8L1, TNF--induced protein 8-like 1), TIPE2 (TNFAIP8L2), and TIPE3 (TNFAIP8L3). TIPE acts as a negative mediator of apoptosis via inhibition of caspase-3 activation and also induces proliferation. TIPE signaling is involved in activation of NF-kappa-B and AP-1 pathways. TIPE-deficient mice have been shown to develop more severe colitis than wild type mice due to TIPE deficiency in non-hematopoietic cells along with higher levels of TNF and IL-6. Given the potential roles of TIPE in regulating these processes and considering the importance of these cytokines and of epithelial injury in the pathophysiology of GVHD, we hypothesized, that TIPE deficiency of allogeneic HCT recipients results in worsened GVHD.

Methods: 9 - 12 week old naive C57BL/6 or TIPE knockout C57BL/6 mice were conditioned with 1000cGy single dose TBI, followed by transplantation of 10 million bone marrow cells and 20 million splenocytes from either syngeneic C57BL/6 or allogeneic BALB/c donors. Animals were monitored for clinical GVHD and survival and analyzed at week 6 after HCT for serum cytokines, T cell expansion, whole spleen cell chimerism and cell proliferation marker.

Results: Allogeneic wild type recipients demonstrated significantly better survival when compared to allogeneic TIPE knockout recipients by week 6 (66.7% vs 40.0%, p =0.024). All syngeneic animals survived. Clinical GVHD scores were significantly higher in TIPE-deficient allogeneic recipients at Day +28 (p=0.0166), +35 (p=0.0349), +42 (p=0.0241) compared to allogeneic wild type recipients. Day +42 serum cytokine analysis revealed increased IL-17A (p=0.0027), TNF (p=0.0396), IL-6 (p=0.0064) levels in TIPE-deficient allogeneic recipients compared to allogeneic control. This was associated with increased splenic CD4+ (p=0.04) but not CD8+ (p= 0.0898) cell expansion when compared to allogeneic control. TIPE-deficient and allogeneic control mice showed no differences in splenic donor T cell chimerism (84.4 versus 89.4, p=0.178), indicating comparable donor T cell engraftment. Ki-67 expression was significantly decreased in epithelial cells of small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls, and a similar nonsignificant trend was seen in the colon as well. Active caspase-3 expression was significantly increased in the small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls and a similar nonsignificant trend was seen in the colon as well. This observation was associated with significantly increased GI tract pathology scores of TIPE-deficient allogeneic recipients compared to allogeneic control. No differences were observed in pathology of lung and liver between TIPE-deficient allogeneic recipients and allogeneic control. Histopathologic analysis of T cell infiltration in small intestine and colon by immunohistochemistry for CD3 antibody revealed no difference between allogeneic groups, suggesting a role of TIPE-deficient nonhematopoietic cells in GVHD pathophysiology.

Conclusion TIPE deficiency in allogeneic C57BL/6 recipients resulted in increased mortality after transplant, which was associated with significant increase in pro-inflammatory cytokines expression and decreased proliferation and increased apoptosis of intestinal epithelial cells, suggesting not only exaggerated inflammatory damage but also decreased epithelial regeneration. Therefore, TIPE seems to be a TNF receptor family member with rather protective function in GVHD gut pathophysiology and overexpression may be a potential future target to prevent or treat this complication after allo HCT.