Aryl hydrocarbon receptor-targeted therapy for CD4+ T cell-mediated idiopathic pneumonia syndrome in mice.

We previously demonstrated that interferon γ (IFN-γ) derived from donor T cells co-opts the indoleamine 2,3-dioxygenase 1 (IDO1) → aryl hydrocarbon receptor (AHR) axis to suppress idiopathic pneumonia syndrome (IPS). Here we report that the dysregulated expression of AP-1 family genes in Ahr-/- lung epithelial cells exacerbated IPS in allogeneic bone marrow transplantation settings. AHR repressed transcription of Jund by preventing STAT1 from binding to its promoter. As a consequence, decreased interleukin-6 impaired the differentiation of CD4+ T cells toward Th17 cells. IFN-γ- and IDO1-independent induction of Ahr expression indicated that the AHR agonist might be a better therapeutic target for IPS than the IDO1 activator. We developed a novel synthetic AHR agonist (referred to here as PB502) that potently inhibits Jund expression. PB502 was highly effective at inducing AHR activation and ameliorating IPS. Notably, PB502 was by far superior to the endogenous AHR ligand, L-kynurenine, in promoting the differentiation of both mouse and human FoxP3+ regulatory CD4+ T cells. Our results suggest that the IDO1-AHR axis in lung epithelial cells is associated with IPS repression. A specific AHR agonist may exhibit therapeutic activity against inflammatory and autoimmune diseases by promoting regulatory T-cell differentiation.

Priming Mesenchymal Stem/Stromal Cells with a Combination of a Low Dose of IFN-γ and Bortezomib Results in Potent Suppression of Pathogenic Th17 Immunity Through the IDO1-AHR Axis.

Preconditioning of mesenchymal stem/stromal cells (MSCs) with the inflammatory cytokine IFN-γ enhances not only their immunosuppressive activity but also their expression of HLA and proinflammatory genes. We hypothesized that prevention of the upregulation of inflammatory cytokines and HLA molecules in IFN-γ-primed MSCs would render these cells more immunosuppressive and less immunogenic. In this study, we discovered the following findings supporting this hypothesis: (1) activated human T cells induced the expression of IDO1 in MSCs via IFN-γ secretion and those MSCs in turn inhibited T-cell proliferation in an AHR-dependent fashion; (2) there was no difference in the expression of IDO1 and HLA-DR in MSCs after priming with a low dose (25 IU/mL) versus a high dose (100 IU/mL) of IFN-γ; (3) the transient addition of bortezomib, a proteasome inhibitor, to culture MSCs after IFN-γ priming decreased the expression of HLA-DR, inflammatory cytokine genes and Vcam1 while increasing the expression of IDO1 and the production of L-kynurenine; finally, MSCs primed with a combination of a low dose of IFN-γ and bortezomib were more effective in inhibiting Th17-mediated idiopathic pneumonia syndrome (IPS) and chronic colitis than unprimed MSCs. Our results suggest that bortezomib significantly eliminates the unfavorable effects of IFN-γ priming of MSCs (increased expression of MHC molecules and inflammatory cytokines and cell aggregation genes) and simultaneously increases their immunosuppressive activity by upregulating IDO1. Taken together, our newly established MSC priming method may contribute to MSC-based cell therapy for inflammatory diseases.

Inhibition of acute lethal pulmonary inflammation by the IDO-AhR pathway.

The lung is a prototypic organ that was evolved to reduce immunopathology during the immune response to potentially hazardous endogenous and exogenous antigens. In this study, we show that donor CD4+ T cells transiently induced expression of indoleamine 2,3-dioxygenase (IDO) in lung parenchyma in an IFN-γ-dependent manner early after allogeneic hematopoietic stem cell transplantation (HSCT). Abrogation of host IDO expression by deletion of the IDO gene or the IFN-γ gene in donor T cells or by FK506 treatment resulted in acute lethal pulmonary inflammation known as idiopathic pneumonia syndrome (IPS). Interestingly, IL-6 strongly induced IDO expression in an IFN-γ-independent manner when deacetylation of STAT3 was inhibited. Accordingly, a histone deacetylase inhibitor (HDACi) could reduce IPS in the state where IFN-γ expression was suppressed by FK506. Finally, l-kynurenine produced by lung epithelial cells and alveolar macrophages during IPS progression suppresses the inflammatory activities of lung epithelial cells and CD4+ T cells through the aryl hydrocarbon receptor pathway. Taken together, our results reveal that IDO is a critical regulator of acute pulmonary inflammation and that regulation of IDO expression by HDACi may be a therapeutic approach for IPS after HSCT.

Induction of indoleamine 2,3-dioxygenase 1 expression in neurons of the central nervous system through inhibition of histone deacetylases blocks the progression of experimental autoimmune encephalomyelitis.

BACKGROUND: A wide array of histone deacetylase (HDAC) inhibitors and aryl hydrocarbon receptor (AHR) agonists commonly arrest experimental autoimmune encephalomyelitis (EAE). However, it is not known whether HDAC inhibition is linked to the AHR signaling pathway in EAE. METHODS: We investigated how the pan-HDAC inhibitor SB939 (pracinostat) exerted immunoregulatory action in the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE mouse model by evaluating changes in of signal transducer and activator of transcription 3 (STAT3) acetylation and the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and AHR in inflamed spinal cords during EAE evolution. We proved the involvement of IDO1 and the AHR in SB939-mediated immunosuppression using Ido1-/- and Ahr-/- mice. RESULTS: Administration with SB939 halted EAE progression, which depended upon IDO1 expression in neurons of the central nervous system (CNS). Our in vitro and in vivo studies demonstrated that SB939 sustained the interleukin-6-induced acetylation of STAT3, resulting in the stable transcriptional activation of Ido1. The therapeutic effect of SB939 also required the AHR, which is expressed mainly in CD4+ T cells and macrophages in CNS disease lesions. Finally, SB939 was shown to markedly reduce the proliferation of CD4+ T cells in inflamed neuronal tissues but not in the spleen or draining lymph nodes. CONCLUSIONS: Overall, our results suggest that IDO1 tryptophan metabolites produced by neuronal cells may act on AHR in pathogenic CD4+ T cells in a paracrine fashion in the CNS and that the specific induction of IDO1 expression in neurons at disease-afflicted sites can be considered a therapeutic approach to block the progression of multiple sclerosis without affecting systemic immunity.

G-CSF-treated donor CD4+ T cells attenuate acute GVHD through a reduction in Th17 cell differentiation.

The immunoregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic peripheral blood cell transplantation (PBCT) have been demonstrated to reduce acute graft-versus-host disease (GVHD). However, the underlying mechanism is still not clear. In this study, we focused on the direct effects of G-CSF on donor CD4(+) T cell responses after transplantation. We observed that lethally irradiated B6D2F1 recipient mice that are transplanted with CD4(+) T cells from G-CSF-treated B6 donors showed mild attenuations in severity and mortality compared with recipients transplanted with PBS-treated CD4(+) T cells. Notably, skin GVHD was significantly reduced, but no such reduction was observed in other organs. Although there was no difference with respect to alloreactive expansion or Foxp3(+) Treg induction, the use of G-CSF-treated CD4(+) T cells significantly reduced the numbers of IL-17-producing and RORγt-expressing cells in the secondary lymphoid organs of allogeneic recipients after transplantation compared with the use of the control cells. Finally, we found that the suppressor of cytokine signaling-3 (SOCS3) expression in G-CSF-treated donor CD4(+) T cells was much higher than that in control CD4(+) T cells. Our results demonstrate that the inhibition of Th17 cell differentiation by SOCS3 induction is associated with the immunoregulatory role of G-CSF in CD4(+) T cell-mediated acute GVHD.

Novel Histone Deacetylase 6 Inhibitor CKD-506 Inhibits NF-κB Signaling in Intestinal Epithelial Cells and Macrophages and Ameliorates Acute and Chronic Murine Colitis.

BACKGROUND: Selective blocking of HDAC6 has become a promising strategy in treating inflammatory bowel disease. CKD-506 is a novel isoform-selective inhibitor of histone deacetylase 6. The present study was performed to evaluate the effect of CKD-506 on the NF-κB signaling pathway in intestinal epithelial cells (IECs) and macrophages and on murine models of acute and chronic colitis. METHODS: RAW264RAW264.7 murine macrophages and COLO 205 human IECs were pretreated with CKD-506 and then stimulated with lipopolysaccharides (LPS). Cytokine expression of TNF-α, interleukin (IL)-6, IL-8, and IL-10 was measured by ELISA. The effect of CKD-506 on NF-κB signaling was evaluated by Western blotting of IκBα phosphorylation/degradation and electrophoretic mobility shift assay. In vivo studies were performed using a dextran sulfate sodium (DSS)-induced acute colitis model, a chronic colitis model in IL-10 knockout mice, and an adoptive transfer model. Colitis was quantified by the disease activity index, colon length, and histopathologic evaluation. RESULTS: CKD-506 suppressed the expression of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α in IECs and macrophages. CKD-506 strongly inhibited IκBα phosphorylation/degradation and the DNA-binding activity of NF-κB. Oral administration of CKD-506 attenuated DSS-induced acute colitis and chronic colitis in IL-10-/- and adoptive transfer models. CKD-506 ameliorated weight loss, disease activity, and histopathologic score in colitis mice and downregulated IκBα phosphorylation and pro-inflammatory cytokine production significantly. CONCLUSIONS: CKD-506 blocked NF-κB signaling in IECs and macrophages and ameliorated experimental acute and chronic murine colitis models, which suggests that CKD-506 is a promising candidate for inflammatory bowel disease treatment as a small molecular medicine.

Therapeutic activity of the histone deacetylase inhibitor SB939 on renal fibrosis.

Fibrosis is the final pathological outcome of many chronic kidney diseases and is quite common. Thus, development of effective anti-fibrotic agents is urgently needed. Although histone deacetylases (HDACs) have been reported to be involved in renal fibrosis, current HDAC inhibitors are unsatisfactory anti-fibrosis drugs. Therefore, more potentially relevant anti-renal fibrosis HDAC inhibitors are needed. We initially found that non-cytotoxic concentrations of SB939 (pracinostat) had strong anti-fibrotic activity, drastically decreasing TGF-ß1-induced alpha smooth muscle actin (α-SMA) expression in the NRK renal fibroblast cell line. Similar anti-fibrotic activity of SB939 on epithelial-to-mesenchymal transition (EMT) was confirmed using the HK-2 human renal proximal tubular epithelial cell line. SB939 inhibited Smad-independent TGF-ß signaling involving the MAPK and PI3K/AKT pathways. To evaluate in vivo anti-fibrotic activity, we administered SB939 in a unilateral ureteric obstruction (UUO) model. SB939 treatment markedly inhibited the accumulation of α-SMA and tissue injury. Inflammatory and pro-fibrotic cytokines in the obstructed kidney were also significantly decreased by SB939 treatment. Our results suggest that SB939 might be a promising therapeutic drug for preventing renal fibrosis.

Effects of topical chondrocyte-derived extracellular matrix treatment on corneal wound healing, following an alkali burn injury.

Numerous treatments have been used in the management of corneal chemical burns; however, no optimal treatment for corneal chemical burns currently exists. The present study investigated the effects of topical chondrocyte-derived extracellular matrix (CD-ECM) treatment on corneal wound healing, using an alkali burn mouse model. Topical treatment with CD-ECM was shown to reduce corneal opacity following an alkali burn. A histological examination observed the presence of regenerated epithelial cells and a small number of inflammatory cells in the corneas of CD-ECM-treated mice. The majority of the inflammatory cells present in the corneas of the phosphate-buffered saline (PBS)-treated mice were neutrophils that expressed matrix metalloproteinase (MMP)-9. The amount of neutrophils was significantly reduced in the corneas of the CD-ECM-treated mice. Furthermore, the expression levels of interleukin (IL)-8 were significantly reduced in the CD-ECM treatment group, but not in the mice that received the PBS treatment. The results of the present study indicate that CD-ECM treatment may accelerate wound healing in a model of alkali burn-induced corneal injury. The therapeutic mechanism may be associated with accelerated reepithelialization and reduced recruitment of MMP-9-expressing neutrophils, through inhibiting the production of IL-8.

Effect of upregulated TLR2 expression from G-CSF-mobilized donor grafts on acute graft-versus-host disease.

Our previous study demonstrated that G-CSF treatment increased the expression of TLR2 in donor grafts; this contributed to rapid engraftment after allogeneic hematopoietic stem cell transplantation (HSCT) in mice. In the current study, we investigated the effects of upregulated TLR2 expression in G-CSF-mobilized donor grafts on acute graft-versus-host disease (GVHD). We found that TLR2 was highly expressed on myeloid cell populations but not T and B cells from the spleens of G-CSF-treated donor mice. After transplantation, the mortality and disease severity in recipients were not significantly different between G-CSF-treated TLR2-/- and wt donor grafts. Although endogenous TLR2 ligand was detected in the serum of both recipients, T cells from TLR2-/- and wt donors have the same ability regarding alloreactivity. Moreover, the blockade of TLR2 signaling in recipients by administering anti-TLR2 blocking antibody after BMT did not lead to a significant difference in acute GVHD compared with control IgG treatment. However, the hematopoietic ability of G-CSF-mobilized lin−c-kit+ HSCs from TLR2-/- donor grafts was lower than that from wt donor grafts. Our results demonstrate that upregulated TLR2 expression in G-CSF-mobilized donor grafts has no effect on acute GVHD, suggesting that TLR2 is a valuable target for increasing HSCT efficiency in order to enhance engraftment without exacerbating acute GVHD.

The tryptophan metabolite 3-hydroxyanthranilic acid suppresses T cell responses by inhibiting dendritic cell activation.

The generation of tryptophan (Trp) metabolites by indoleamine 2,3-dioxygenase (IDO) is an effective mechanism for T cell suppression. However, the effect of Trp metabolites on dendritic cells (DCs) remains unclear. Here, we investigated whether the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) directly inhibits DC activation and is responsible for T cell suppression. We found that 3-HAA treatment significantly reduced IL-12, IL-6, and TNF-α production in bone marrow-derived DCs (BMDCs) stimulated with LPS. Maturation markers CD40, CD80, CD86, and I-A were also significantly reduced. Moreover, treatment with 3-HAA decreased the ability of DCs to stimulate T cell activation and differentiation in vitro and in vivo. Finally, we observed that phospho-JNK and phospho-38 levels were reduced in 3-HAA-treated DC2.4 cells and BMDCs. These results suggest that the tryptophan metabolite 3-HAA suppresses T cell responses by inhibiting DC activation.