Intestinal Epithelial-Derived IL-34 Regulates Macrophage Polarization in an Apolipoprotein E Dependent Manner and Mitigates the Severity of Graft Versus Host Disease

Acute graft versus host disease (GVHD) of the gastrointestinal (GI) tract is a major complication of allogeneic bone marrow transplantation. While donor T cells are the proximate trigger of GVHD, innate cells also contribute to disease pathogenesis, although the role of these cell populations is not as well defined. Macrophages, for example, are known contributors in the pathophysiology of chronic GVHD, but their role in acute GVHD is not well delineated. Fundamentally, macrophages are dependent upon exposure to m-CSF and IL-34 which serve as critical trophic factors necessary for their development through binding to the CSF-1R. IL-34 is a more recently discovered cytokine produced by macrophages, but whose role in GVHD pathogenesis is unknown. To examine this question, we employed a murine bone marrow transplantation model (B10.BR [H-2k]®B6[H-2b]) and observed that that there was no difference in GVHD-induced lethality when recipients were reconstituted with marrow grafts from wild type (WT) or IL-34^-/- donors. In contrast, recipient IL-34^-/- animals had significantly worse survival compared to WT mice indicating that host IL-34 expression protected animals from lethal GVHD. We observed no difference in the absolute number of donor macrophages in the colons of WT versus IL-34^-/- mice; however, IL-34^-/- recipient mice had a significant increase in the number of inflammatory Ly6C⁺ macrophages, and a corresponding decrease in Ly6C^- cells. Single cell RNA sequence (scRNAseq) analysis of donor macrophages from the colons of WT and IL-34^-/- recipients revealed that macrophages from WT animals had increased expression of anti-inflammatory genes (i.e., CD83, ApoE, Selenop, Lyz1 and Mrc1), whereas macrophages from IL-34^-/- recipients had increased expression of pro-inflammatory genes (i.e., Ifitm1, S100A8, S100A9 and GOs2), indicating that absence of host IL-34 skewed donor macrophages towards an inflammatory phenotype. Since the CSF-1R is not expressed on T cells, we sought to determine the effect of IL-34/CSF-1R signaling on proinflammatory T cells in GVHD target organs. scRNAseq analysis of colonic CD4+ T cells revealed that Csf2 (GM-CSF) was the most differentially expressed gene in IL-34^-/- CD4+ T cells. Functionally, this was confirmed by the observation of a significantly increased number of CD4⁺ GM-CSF+ T cells in the colons of these mice. Notably, there was no difference in proinflammatory CD8⁺ T cells, nor any effect on T cell phenotype in the liver or lung, demonstrating that the mitigating effect of host IL-34 expression was confined to the GI tract. To determine the cellular source of IL-34 production, we employed IL-34 LacZ reporter mice and showed that β-Gal co-localized with the epithelial marker, E-cadherin, indicating IL-34 is produced primarily by intestinal epithelial cells. Additional studies using chimeric mice in which IL-34 was restricted to either the hematopoietic or non-hematopoietic compartment revealed that absence of IL-34 in the non-hematopoietic compartment phenocopied findings observed with global IL-34^-/- animals, confirming that intestinal epithelial cells were the primary source of this cytokine. The administration of exogenous IL-34 prolonged survival, abrogated pathological damage in the colon and resulted in a corresponding decrease in pro-inflammatory macrophages and T cells, demonstrating this pathway could be pharmacologically targeted. Re-analysis of the scRNAseq data on donor macrophages from the colons of WT and IL-34^-/- mice revealed that apolipoprotein E (Apoe) was the most differentially expressed gene in macrophages from WT GVHD animals. To confirm a functional role for ApoE, mice were transplanted with T cell depleted (TCD) BM from either WT or ApoE^-/- mice and treated with IL-34. These studies revealed that the protective effect conferred by IL-34 was abrogated in animals that received TCD ApoE^-/- BM grafts, indicating that ApoE expression in donor macrophages was necessary for agonist driven IL-34-mediated regulation of GVHD. Finally, we confirmed these studies have translational relevance by demonstrating IL-34 and Apoe have increased expression in intestinal epithelial cells and macrophages, respectively, in the colons of humans with GVHD. Thus, these results identify IL-34 as a novel intestinal epithelial cell-derived cytokine that regulates macrophage polarization and mitigates GVHD in an ApoE-dependent manner.