A Bhlhe40/GM-CSF Axis Potentiates Gastrointestinal Tract Inflammation during Acute Graft Versus Host Disease
Authors: Piper Clint , Zhou Vivan , Edelson Brian T. , Taneja Reshma , Drobyski William R. 
Published: October 2019

Abstract
Damage to the gastrointestinal tract is the major cause of morbidity during acute graft-versus-host disease (GVHD). While T cells are the proximate drivers of GVHD, disease induction and amplification rely on crosstalk between the innate and adaptive arms of the immune system. The cellular and cytokine networks which mediate this interplay, however, are not well understood. We previously identified a colitogenic CD4+ IL-23R+ CD11c+ T cell population that possesses an innate-like gene expression signature, indicating that these cells appear to be positioned at the interface of the innate and adaptive arms of the immune system. Notably, we also observed that these cells had increased expression of bhlhe40 which is a transcription factor that has been shown to regulate the production of GM-CSF. Given the well characterized ability of GM-CSF to activate myeloid cell populations in autoimmunity, we sought to define the role of this transcription factor and cytokine as a potential bridge between innate and adaptive immunity in GVHD. Using a well-defined murine GVHD model [C57BL/6 (H-2b)→Balb/c (H-2d)], we observed that mice transplanted with Rag-1-/- bone marrow (BM) and CD4+ bhlhe40-/- T cells were completely protected from GVHD, whereas animals transplanted with Rag-1-/- BM and wild type (WT) CD4+ T cells uniformly developed lethal disease. Further analysis revealed that CD4+ bhlhe40-/- T cells produced less GM-CSF and more IL-10 than their WT counterparts, and had preferentially less pathological damage in the colon. To examine the specific role of GM-CSF, we employed the same GVHD model along with a corresponding syngeneic control (B6→B6.PL). We observed robust GM-CSF production in allogeneic, but not syngeneic, recipients in all GVHD target tissues, but most prominently in the colon. This was largely attributable to donor-derived CD4+ T cells, as there was little GM-CSF produced by CD8+ T cells. Notably, whereas the vast majority (~80%) of these cells in the lung and liver also produced IFN-γ, ~50% of GM-CSF-expressing CD4+ T cells in the colon only produced GM-CSF, suggesting that these cells might represent a separate CD4+ T cell lineage. In that regard, antibody blockade of IL-6, IL-23 and IL-27 had no effect on the frequency of CD4+ GM-CSF+ T cells, indicating that the development of these cells was not regulated by cytokines affecting TH1 and TH17 differentiation. To define the functional significance of donor T cell-derived GM-CSF, recipients were transplanted with BM or BM plus splenocytes from WT or GM-CSF-/- animals. Recipients of GM-CSF-/- grafts had significantly increased survival when compared to WT controls. Furthermore, histological analysis demonstrated a significant reduction in pathology in the colon of animals that received GM-CSF-/- grafts, as well as a decrease in infiltrating TH1 cells, whereas there was no difference in pathological damage in the lung or liver. A similar outcome was observed in complementary experiments in which recipient animals that were treated with an anti-GM-CSF antibody had significantly increased survival compared to mice treated with an isotype control antibody. To confirm a role for GM-CSF signaling in CD4+ T cells, Balb/c recipients were transplanted with Rag-1-/- BM alone or together with purified CD4+ T cells from WT or GM-CSF-/- mice. Mice that received CD4+ GM-CSF-/- T cells had a significant increase in survival compared to those that received WT CD4+ T cells, confirming a proinflammatory role for GM-CSF production by donor CD4+ T cells. Given that GM-CSF acts on a diverse subset of innate immune cells, we then examined which myeloid cell subsets were responsive to GM-CSF two weeks post-transplantation when donor APCs have repopulated the APC compartment. Using established markers for macrophages, neutrophils, and dendritic cells, we observed no difference in the number of donor macrophages or neutrophils between groups. However, there was a significant reduction in dendritic cells (DCs) in the colon of mice receiving CD4+ GM-CSF-/- T cells, and donor-derived DCs were virtually absent from the mesenteric lymph nodes, indicating that GM-CSF facilitates the accumulation of DCs in the GI tract and associated lymphoid tissue during GVHD. Collectively, these studies demonstrate that a CD4+ T cell-intrinsic bhlhe40/GM-CSF axis potentiates gastrointestinal inflammation during GVHD by promoting inflammatory cytokine production and DC recruitment.

Disclosures
No relevant conflicts of interest to declare.