Abstract
The initiation of graft-versus-host disease (GVHD) is dependent upon donor T cell recognition of host alloantigens presented within the context of host antigen presenting cells (APCs). Eventually host APCs are replaced by donor APCs, however the role of the latter in the propagation of GVHD remains largely unknown. Recently, interleukin 23 (IL-23) has been identified as a cytokine that is secreted exclusively by APCs, and has been shown to play a pivotal role in several inflammatory disorders. The purpose of this study therefore was to address whether secretion of IL-23 by donor APCs played a role in the pathophysiology of GVHD. Transplantation of bone marrow and spleen cells from IL-23 deficient C57BL/6 (B6) (H-2b) donors into lethally irradiated MHC-incompatible Balb/c (H-2d) or FVB (H-2q) mice resulted in significantly less weight loss and proinflammatory cytokine production, as well as improved immune reconstitution, when compared to animals transplanted with marrow grafts from normal B6 mice. Histological examination of GVHD target tissues revealed that, in the absence of donor APC-derived IL-23 secretion, there was a selective and profound reduction in pathological damage in the colon, without any corresponding reduction in either the lung or liver. This was accompanied by a marked decrease in lipopolysaccharide (LPS) and proinflammatory cytokine production (IFN-γ, G-CSF, TNF-α, IL-1β and IL-6) in the colon microenvironment. To further investigate the preferential protection observed in the colon, we examined IL-23 mRNA levels in the spleen, liver and colon to determine whether there was differential gene expression during GVHD. In comparison to normal nontransplanted Balb/c mice, there was a selective increase in IL-23 (3-fold) mRNA levels in the colon of GVHD animals, but there was no difference between normal and GVHD mice in the spleen or liver. Examination of IL-23 receptor mRNA levels in these same tissues revealed a marked increase only in the colon of GVHD animals. To define the donor APC populations in the colon responsible for secretion of IL-23, CD11c+ CD11b+ dendritic and CD11b+ CD11c− myeloid/monocytic cells were examined for expression of IL-23 mRNA. Levels were significantly increased (600-fold) in CD11c+ CD11b+ cells derived from the colon as opposed to the spleen of GVHD mice where IL-23 expression was undetectable. Similarly, gene expression of IL-23 was 20-fold higher in colon versus splenic-derived CD11b+ CD11c− cells. Expression of the IL-23R was most dramatically increased (~1000-fold) in purified colon-derived CD4+ T cells whereas CD4+ T cells isolated from the spleen or liver of these same mice had negligible expression, suggesting that APC-derived IL-23 acted primarily through donor-derived CD4+ T cells. Since IL-23 induces secretion of IL-17 and IFN-γ by memory CD4+ T cells, we performed studies to determine if pathological damage was mediate by either of these cytokines. Transplantation with marrow grafts from IL-17-deficient donors had no protective effect on either overall or colon-specific pathology, providing evidence that the proinflammatory effects of IL-23 were independent of IL-17. Conversely, transplantation with IFN-γ−/− marrow grafts resulted in a significant reduction in colonic pathology compared to recipients of wild type grafts and was associated with a marked reduction in LPS and IL-23 levels as well as proinflammatory cytokine production demonstrating that the inflammatory effects of IL-23 in the colon were mediated, in large measure, through donor-derived IFN-γ. Finally, we observed that TLR-4 gene expression was selectively augmented in the colon when compared to the spleen and liver. The importance of the LPS/TLR4 signaling pathway was further confirmed by studies demonstrating that mice transplanted with TLR4- deficient marrow grafts had significantly reduced IL-23 and LPS levels as well as pathological damage in the colon. In summary, these data demonstrate that IL-23 has a unique and selective role in the induction of colonic inflammation during acute GVHD and serves as a critical mediator linking conditioning regimen-induced mucosal injury and LPS translocation to subsequent proinflammatory cytokine production and GVHD-associated pathological damage. These findings define a novel organ-specific role for IL-23 in the pathophysiology of GVHD and identify IL-23 as a therapeutic target for the prevention of this disease.
TGF-β1 inhibition of IFN-γ-induced signaling and Th1 gene expression in CD4+ T cells is Smad3 independent but MAP kinase dependent
