Host Interleukin 6 and Indoleamine 2,3 Dioxygenase Regulate Inflammation in the Brain during Graft Versus Host Disease

Graft versus host disease (GVHD) commonly induces pathological damage in peripheral target organs such as the skin, liver and gastrointestinal tract leading to well characterized organ-specific clinical manifestations. A number of studies, however, have shown that patients with GVHD can also have behavioral and mood alterations that can affect overall cognitive function and lead to significant impairments in quality of life. The extent to which GVHD contributes to cognitive dysfunction and induces inflammation within the central nervous system (CNS), however, has not been critically examined. To address this question, we conducted studies using two well-defined murine GVHD models [C57BL/6(H-2^b)→Balb/c (H-2^d) and B10.BR(H-2^k)→B6 (H-2^b)]. We observed that there was a significant increase in the number of donor-derived CD4⁺ and CD8⁺ T cells in the brains of GVHD recipients early (days 7 -14) and late (day 42) post transplantation compared to BM controls. Histological studies revealed activated microglial cells and CD3⁺ T cell infiltration in the periventricular regions of brains in GVHD recipients that were not present in BM animals. Real time q-PCR analysis also demonstrated significant increases in IFN-γ, TNF-α, and IL-6 mRNA expression indicative of a proinflammatory state. Notably, GVHD animals exhibited behavioral changes in the forced swim test and elevated plus maze which are validated assays of stress coping and anxiety, respectively. Since IL-6, in particular, plays a pivotal role in GVHD pathogenesis in murine models and humans, we examined whether blockade of IL-6 signaling altered neuroinflammation. Animals treated with an anti-IL-6R antibody had a significant reduction in the number of donor-derived CD4⁺ and CD8⁺T cells in the brain compared to isotype control-treated mice. Anti-IL-6R treatment of GVHD mice also resulted in significant reductions in IFN-γ, TNF-α, and IL-6 mRNA and normalized behavior in the forced swim test, indicative of a decreased inflammatory response. Since IL-6 is produced by a wide variety of cells, including microglial and T cells, both donor and recipient cells have the potential to modulate GVHD severity within the CNS. To define whether donor or host IL-6 production was most critical for inducing neuroinflammation, experiments were conducted employing IL-6-/- mice as either donors or recipients. Whereas the absence of IL-6 in donor-derived cells had no impact on the degree of inflammation within the CNS, recipient animals that lacked IL-6 had a significant decrease in the number of donor-derived T cells which accumulated in the brain as well as a marked reduction in inflammatory cytokines, indicating that host IL-6 production was critical. To define the downstream pathways of IL-6-mediated CNS inflammation, we examined the role of indoleamine 2,3-dioxygenase (IDO) since IL-6 has been shown to upregulate IDO-1 expression under inflammatory conditions. We observed that IDO-1 mRNA levels were significantly increased in the brains of GVHD animals, and that blockade of IL-6 signaling resulted in a marked decrease in IDO mRNA levels. Additionally, transplantation studies using IDO-/- mice revealed that host, but not donor, IDO production was required for maximal inflammatory effects. Serotoninergic projections to the prefrontal cortex (PFC), in particular, are sensitive to inflammation and contribute to stress coping behavior. Therefore, to further interrogate this pathway, we performed quantitative mass spectrometry of brain extracts from the PFC. We found that tryptophan and 5HT concentrations were not different between BM and GVHD groups. However, there was an increase in the IDO product, kynurenic acid, in GVHD recipients consistent with an increase in brain IDO expression. To provide additional support for the premise that IL-6 effects were mediated through the IDO pathway, recipient mice were treated with either 1-methyltryptophan (1-MT), a completive inhibitor of IDO, or a vehicle control. GVHD mice treated with 1-MT had decreased accumulation of T cells in the brain and normal behavior in the forced swim test, demonstrating that inhibition of IDO abrogated CNS inflammation and behavioral changes in the presence of intact IL-6 signaling. In summary, these studies demonstrate that host IL-6 and IDO regulate inflammation and adversely impact behavioral function within the brain during GVHD through the tryptophan metabolic pathway.