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. Author manuscript; available in PMC: 2021 May 21.
Published in final edited form as: Transplantation. 2020 Dec;104(12):2507–2515. doi: 10.1097/TP.0000000000003383

The Prolyl Hydroxylase Inhibitor Dimethyl Oxalyl Glycine Decreases Early Gastrointestinal GVHD in Experimental Allogeneic Hematopoietic Cell Transplantation

Senthilnathan Palaniyandi 1,#, Reena Kumari 1,#, Sabarinath Venniyil Radhakrishnan 2, Ethan Strattan 1,3, Natalya Hakim 4, Reinhold Munker 1, Melissa V Kesler 4, Gerhard C Hildebrandt 1,3
PMCID: PMC8139022  NIHMSID: NIHMS1699322  PMID: 32639407

Abstract

Background.

Prolyl hydroxylase inhibitors (PHI) promote stabilization of hypoxia-inducible factor-1 alpha and affect signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection led us to test the effect of the PHI dimethyl oxalyl glycine (DMOG) in the pathophysiology of graft versus host disease (GVHD).

Methods.

Acute GVHD was induced in lethally irradiated BALB/c mice. DMOG was administered intraperitoneally on alternate days for the first 2-weeks posttransplant, and then twice a week till day +50, while controls received vehicle only. Animals were monitored for clinical GVHD and analyzed at day +7 and at day +50.

Results.

DMOG treatment of allogeneic recipients improved survival by day +50, which was associated with decreased early gut injury and serum tumor necrosis factor-α compared with allogeneic controls. DMOG treatment of allogeneic recipients resulted in increased hypoxia-inducible factor-1 alpha expression and reduced apoptosis in the terminal ileum via Fas-associated protein with death domain protein repression along with decreased T-cell infiltration. Reduced pathology in colon after DMOG treatment associates with intestinal epithelium integrity and reduced damage caused by diminished recruitment of neutrophils.

Conclusions.

Taken together, we show protective effects of DMOG on early gut GVHD and improved survival in a model of allogeneic hematopoietic cell transplantation, providing the rationale for further evaluation of PHIs, in the prevention and treatment of acute GVHD.

INTRODUCTION

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for a variety of hematological malignancies. The number of allo-HCTs is increasing, and currently there are >25 000 procedures carried out annually. Its benefits are countered by the risk of graft versus host disease (GVHD), which may result in significant morbidity and mortality.1 The pathophysiology of acute GVHD can be hypothesized in 3 sequential phases: the phase of conditioning therapy-related injury and inflammatory cytokine storm; the phase of donor T-cell activation involving interactions between host antigen-presenting cells and donor T cells and the cellular effector phase. Primary organs affected by this alloreactive T-cell response are the intestine, liver, and skin, hematopoietic system and lung. The initiation of these events involves injury to the gut, leading to a cascade of inflammatory events through translocation of pathogen-associated molecular patterns and damage-associated molecular patterns, then propagating donor T-cell activation.2 Acute GVHD accounts for 15%–30% of deaths that occur following allo-HCT and is a major cause of morbidity in up to 50%–60% of transplant recipients.1 However, alloreactive T-cell responses are also beneficial exerting antileukemic effects reducing the risk of relapse (graft versus leukemia effects).

Hypoxia-inducible factors (HIFs) are a basic mechanism protecting the organism against hypoxia. A significant role of HIF-1 was suggested in inflammatory bowel disease (IBD).3 HIF-1 provides barrier protection in the colonic epithelium following mucosal insult. The HIFs are primarily regulated by prolyl hydroxylases at the level of protein stability. These prolyl hydroxylase domain (PHD) proteins are members of a broader family of nonheme, iron-, and 2-oxoglutarate-dependent dioxygenases.4

Dimethyloxalyl glycine (DMOG) is a cell permeable prolyl-4-hydroxylase inhibitor and is specific to the 2-oxoglutarate (2-OG) dioxygenase family, including prolyl hydroxylases (PHD1, PHD2, PHD3 1) and factor inhibiting HIF (FIH). PHDs control the degradation of HIF through proline hydroxylation, whereas FIH-dependent asparagine hydroxylation regulates interactions with CREB-binding protein/p300 involved in fine tuning HIF activity.5

Thus, DMOG may result in the transcriptional induction of a subset of genes responsive to PHD and FIH inhibition.6 DMOG stabilizes and transactivates HIF-1α leading to increased endogenous HIF-1α levels. HIF-1α is involved in the antiinflammatory effects of hypoxia-signaling through a transcriptional program, and it has been shown that pharmacologic activators of HIFs provide tissue protection in murine models of colitis.7

Identifying effective and specific inhibitors of critical immune signaling events in the early and later stages of GVHD development is crucial to the success of allo-HCT. Both novel preventive and therapeutic approaches are needed. Given the role of HIF-1α in molecular hypoxic response, inflammation, and tissue injury we hypothesized that use of the prolyl hydroxylase inhibitor (PHI) DMOG reduces organ injury after murine allo-HCT, resulting in decreased GVHD and improved overall survival.

MATERIALS AND METHODS

Mice, Induction of GVHD and DMOG Treatment

BALB/c (H-2d) and C57BL/6(B6, H-2b) mice were purchased from The Jackson Laboratories, United States, and housed in micro isolator cages in the specific pathogen free facility. Mice were cared and treated with accordance with the Institutional Animal Care and Use Committee (IACUC) of the Louisiana State University Health Sciences Center, Shreveport, and University of Kentucky. The BALB/c recipient mice were conditioned with 750cGy single-dose total body radiation (TBI) using a Cesium source irradiator, followed by intravenous infusion of 5 × 106 bone marrow cells and 4 × 106 splenocytes from either syngeneic BALB/c or allogeneic C57BL/6 donors. Mice at 9–12 weeks of age were used for the experiments. DMOG (CAYMAN Chemical Company, Michigan) dissolved in phosphate buffer saline (PBS) was given to both syngeneic and allogeneic recipients at a dose of 8 mg per mouse intraperitoneally on alternate days for the first 2-week posttransplant and then twice a week until day +50, while syngeneic and allogeneic controls received PBS only. At day +7 and at day +50, the recipients were analyzed for GVHD target organ pathology, cytokine levels, and immune cell phenotypes.

Assessment of aGVHD

Recipient mice were monitored daily for survival, and clinical aGVHD was assessed weekly. Mice were scored for clinical aGVHD by assessment of 5 parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity. Individual mice from coded cages received a score of 0–2 for each criteria (maximum score of 10) that was used as an index of severity and progression of disease.8

Serum Cytokine Analysis

The levels of serum cytokines were determined using the Cytometric Bead Array Mouse Cytokine kit (BD Biosciences, MD). Mouse cytokine-specific bead set and standards were implemented as per the manufacturer’s instructions. The fluorescence produced by the beads was measured using FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed with the accompanying software.

Serum and Tissue Chemokine Analysis

Tissues were harvested and snap-frozen. Supernatant of homogenized tissue was prepared as described before.9 Total protein concentration in the supernatant was quantified by using bicinchoninic acid assay (Thermo Scientific, Rockford, IL) to allow for cytokine and chemokine concentration normalization. Tissue homogenates and serum samples were subjected for chemokine quantitation using LEGENDplex mouse proinflammatory chemokine panel (13-plex) assay kit, which analyzes RANTES (CCL5), Eotaxin (CCL11), TARC (CCL17), KC (CXCL1), MIP-3α (CCL20), MIG (CXCL9), IP-10 (CXCL10), BLC (CXCL13), LIX (CXCL5), MDC (CCL22), MCP-1 (CCL2), MIP-1α (CCL3), and MIP-1β (CCL4).

Histopathology and Immunohistochemical Analysis

GVHD target organ pathology in liver, lung, terminal ileum, and colon tissues was assessed by histological examination. Paraffin-embedded tissues were sectioned, deparaffinized, and stained with hematoxylin and eosin (H&E). Histological examination and GVHD damage assessment were independently carried out by 2 pathologists in a completely blinded fashion. Intestinal GVHD was scored on the basis of crypt apoptosis (0, rare to none; 1, occasional apoptotic bodies per 10 crypts; 2, few apoptotic bodies per 10 crypts; 3, the majority of crypts contain an apoptotic body; 4, the majority of crypts contain >1 apoptotic body; and inflammation (0, none; 1, mild; 2, moderate; 3, severe, without ulceration; 4, severe, with ulceration).8,10,11

For immunohistochemistry (IHC), formalin-fixed paraffin sections were deparaffinized in xylene and dehydrated in ethanol series (100%, 95%) and subjected to antigen retrieval in a rice-steam cooker using Target retrieval solution (Dako, Carpinteria, CA) after rehydration in PBS. After retrieval sections were incubated with respective primary antibodies for HIF-1α (5 μg/mL) (Abcam), CD3e (1:200) (Cell Signaling Technology), phospho FAS–associated protein with death domain (FADD) pSer191 (1:50 dilution) (Thermofisher scientific) or myeloperoxidase (MPO) (0.75 μg/mL) (Abcam).

For zonula occludens-1 (ZO-1) IHC, all sections after deparaffinization were subjected to antigen retrieval for 10 minutes using protease from Streptomyces gruseus, type XIV, 1.5 mg/mL (Sigma-Aldrich) and washed in PBS followed by incubation with ZO-1 antibody (3.5 μg/mL) (Thermofisher scientific).12

After incubation with primary antibody, slides were followed by incubation with rabbit horseradish peroxidase-labeled polymer (DakoEnVision+ system-horseradish peroxidase labeled polymer) or goat antimouse IgG H&L (alkaline phosphatase) (Abcam, Cambridge, MA) depending on the primary antibody used. The sections were developed using either with Dab buffer (Dako, Carpinteria, CA) or StayGreen/AP Plus (Abcam, Cambridge, MA) depending on the conjugate on the secondary antibody. Pictures were captured using Nikon eclipse 55i microscope, and IHC data were analyzed through ImageJ software. For TUNEL assay, deparaffinized sections were subjected to chromogenic TUNEL assay and slides were scanned through Aperio software, and data were analyzed in Aperio software using Nuclear MC algorithm.

Isolation and Culture of Peritoneal Macrophages With Lipopolysaccharides and DMOG

The peritoneal cavity was lavaged with 3 mL ice-cold PBS and 2 mL of air. The resulting cells were washed and resuspended in Dulbecco's Modified Eagle Medium supplemented with 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in a humidified atmosphere at 37°C and 5% CO2 for 2 hours, and nonadherent cells were removed. The remaining adherent cells were treated with lipopolysaccharides (LPS) (100 ng/mL) with or without DMOG (1 mmol/L) presence and cultured further for 4 hours. After incubation, supernatants were collected and subjected to ELISA for tumor necrosis factor (TNF) (BD Biosciences Pharmingen, San Diego, CA).13,14

Statistical Analysis

Experimental data are expressed as means ± SD. Statistical comparisons between groups were made using either student’s t test or 1-way ANOVA. A P ≤ 0.05 was considered statistically significant. Survival curves were analyzed with Log-rank (Mantel-Cox) test.

RESULTS

DMOG Administration Ameliorates Early Gastrointestinal Injury and Early Acute GVHD After allo-HCT

To determine the effect of DMOG on the development of acute GVHD, we used a well-characterized murine mismatch HCT model. Acute GVHD was induced by intravenous injection of bone marrow and splenocytes from C57BL/6 into lethally total body irradiated BALB/c mice. By day 50 after HCT, 22% of allogeneic control recipients compared with 59% of DMOG-treated allogeneic recipients were alive (Figure 1A; P < 0.05). None of the syngeneic recipients had died.

FIGURE 1.

FIGURE 1.

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DMOG administration ameliorates early gastrointestinal injury and early acute GVHD after allo-HCT. A, Survival at d +50 after HCT from 2 independent experiments. Animals underwent transplantation as described in Materials and Methods. DMOG dissolved in PBS was given to both syngeneic and allogeneic recipients at 8 mg per mice intraperitoneally on alternate days for the first 2-wk posttransplant and then twice a week till d +50, while syngeneic and allogeneic controls received PBS only (sample size at d +50: syngeneic control: n = 7; DMOG-treated syngeneic control: n =5; allogeneic control: n = 18; DMOG-treated allogeneic: n = 17). Survival curves were analyzed with Log-rank (Mantel-Cox) test. B–E, Pathology of the terminal ileum, colon, lung, and liver by H&E staining at d 7 after HCT using scoring systems as described in Materials and Methods). F–H, Cytokine levels in serum at d +7. Data are from 2 independent experiments. Sample size at d +7: syngeneic control: n = 6; DMOG-treated syngeneic control: n = 6; allogeneic control: n = 12; DMOG-treated allogeneic: n = 12. I, TNF production from LPS (100 ng/mL) stimulated peritoneal macrophages with and without DMOG (1 mmol/L) treatment. Results shown as mean ± SD, from 1 experiment of 2 comparable experiments. Statistical comparisons between groups were made using either Student’s t test or 1-way ANOVA. A P ≤ 0.05 was considered statistically significant. J, Representative pictures of histological analysis of disease severity in ileum and colon at d +7. DMOG, Dimethyl oxalyl glycine; GVHD, Graft versus host disease; HCT, hematopoietic cell transplant; LPS, lipopolysaccharides; PBS, Phosphate buffer saline; TNF, tumor necrosis factor.

Given the strong role for conditioning-related intestinal damage in initiating and intensifying systemic GVHD and considering the protective effects of DMOG on the gastrointestinal tract in a dextran sulphate sodium colitis model,7 we assessed GVHD target organ pathology at day +7. A significant reduction in pathologic damage of terminal ileum and colon was observed in allogeneic DMOG-treated recipients when compared with allogeneic controls (Figure 1B, C, and J; P < 0.05), whereas histopathologic changes in lung and liver were not statistically different (Figure 1D and E). No significant differences were seen in GVHD clinical scores (data not shown). Systemic proinflammatory cytokine serum levels showed a reduction in TNF-α, whereas no significant changes were observed for interferon gamma (IFN-Υ) and interleukin 6 (IL-6) in the DMOG-treated allogeneic group compared with allogeneic controls (Figure 1F-H). Intestinal macrophages play an important role in propagating TBI-induced inflammation and GVHD of the intestinal tract and are significant sources of TNF.11,15 We observed significant decrease in TNF production from LPS-stimulated peritoneal macrophages in the presence of DMOG (Figure 1I).

We then assessed GVHD organ pathology in surviving animals at day +50, a time point when the early cytokine storm has mostly subsided and tissue-infiltrating effector T cells are the driving cellular factors for GVHD pathology. At this later time point, no significant changes were seen in pathology (data not shown).

DMOG Treatment in Allogeneic Recipients Stabilizes Intestinal Epithelial HIF-1α Protein and Decreases T-cell Infiltration and Reduces Apoptosis in the Terminal Ileum

We next studied how DMOG effects HIF expression and tissue injury. We analyzed HIF-1α expression in the terminal ileum and colon by IHC. Posttransplant treatment with DMOG versus PBS resulted in increased HIF-1α in terminal ileal sections at day +7 (P < 0.05) as well as day +50 (P < 0.05; Figure 2A and B), whereas no significant difference in HIF-1α protein expression was found in the colon at either time point (data not shown). NF-kB is induced following hypoxia.16 Analysis of NF-kB and HIF-2 α showed no difference (data not shown).

FIGURE 2.

FIGURE 2.

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DMOG treatment in allogeneic recipients stabilizes intestinal epithelial HIF-1α protein alongside decreased T-cell infiltration and results in reduced apoptosis at d +7 in the terminal ileum. A, Expression of HIF-1α by IHC in small intestine at d +7 and d +50. B, Representative pictures of HIF-1α stained (green signal, shown by blue arrowhead) and CD3 stained (brown signal, shown by red arrowhead) small intestine sections of allogeneic control and DMOG-treated allogeneic mice at d +7. C, T-cell infiltration by CD3 by IHC in small intestine at +7 and d +50. D, Expression of FADD pSer191-a by IHC in small intestine sections at +7 and d +50. E, Representative pictures of FADD expression by IHC in terminal ileum of allogeneic control and DMOG-treated allogeneic mice at d +7. F, Apoptosis by chromogenic TUNEL assay in the terminal ileum sections at d +7 and d +50. G, Representative pictures of chromogenic TUNEL assay in terminal ileum of allogeneic control and DMOG-treated allogeneic mice at d +7. IHC data were analyzed through imageJ software and the Y axis in A, C, and D indicates % of positive area for respective antibody. The Y axis in F indicates % of positive nuclei for TUNEL staining. Sample size at d +7 allogeneic control: n = 6; DMOG-treated allogeneic: n = 6; sample size at d +50, allogeneic control: n = 4; DMOG-treated allogeneic: n = 6. Results shown as mean ± SD. Statistical comparisons between groups were made using either Student’s t test or 1-way ANOVA. A P ≤ 0.05 was considered statistically significant. DMOG, dimethyl oxalyl glycine; FADD, FAS-associated protein with death domain; HIF-1α, hypoxia-inducible factor-1 alpha; IHC, immunohistochemistry.

Next, we analyzed infiltration of CD3+T cells in the small intestine (terminal ileum) by IHC. We observed a significant decrease in T-cell infiltration at day +7 and +50 as well in DMOG-treated allo recipients compared with allo recipients (Figure 2B and C). No difference was seen for T-cell expansion in the spleen (data not shown) and T-cell infiltration in colon (data not shown). Next, we determined the extent of apoptosis in the terminal ileum as a hallmark of GVHD injury. We measured FADD protein expression due to its pivotal role in TNF-mediated apoptosis. FADD also serves as one of the potential targets of DMOG.17 Hindryckx et al17 showed that HIF-1α binds effectively to the endogenous FADD promoter and has a potential role in transcriptional repression of FADD. HIF-1α directly regulates promoter activity of FADD and controls repression of FADD during hypoxia. We also observed reduced FADD expression in terminal ileum of allogeneic DMOG-treated recipients at days +7 and +50 (Figure 2D and E; P < 0.05), suggesting that DMOG represses intestinal epithelial FADD expression via HIF-1α stabilization in terminal ileum, which makes the cells less susceptible to TNF-α-mediated apoptosis. To further confirm the effect of DMOG on apoptosis via FADD repression, we measured the apoptosis in the terminal ileum at days +7 and +50 through chromogenic TUNEL assay and observed a decrease in apoptosis at day +7 (no difference at day +50; Figure 2F and G).

DMOG Resulted in Reduced Proinflammatory Chemokine Levels in Serum and in Both Ileum and Colon

Chemokine analysis revealed significantly reduced levels of MCP-1 and KC in the serum (Figure 3A), of RANTES, MIP-1α, MIP-1β, and MCP-1 in the ileum (Figure 3B), and of KC in colon lysate of DMOG-treated recipients compared with allo controls (Figure 3C). No statistically significant differences were seen for other chemokines in respective organs.

FIGURE 3.

FIGURE 3.

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DMOG results in reduced proinflammatory chemokine levels in serum and in both ileum and colon. A, Expression of proinflammatory chemokine in serum (B) ileum and (C) colon tissue homogenate measured by LEGENDplex mouse proinflammatory chemokine panel assay kit as described in Material and Methods. Sample size at d +7 syn control: 3; syn DMOG: 3; allogeneic control: n = 6; DMOG-treated allogeneic: n = 6. (results shown as mean ± SD). Statistical comparisons between groups were made using student’s t test. A P ≤ 0.05 was considered statistically significant. DMOG, dimethyl oxalyl glycine.

DMOG Treatment Protects the Intestinal Epithelium

DMOG is known to preserve the barrier function in DSS colitis18 by regulating ZO-1 expression. To see the effect of DMOG on intestinal integrity, we analyzed the expression of a key regulator of paracellular permeability, ZO-1 in ileum and colon by IHC. We observed significant loss of ZO-1 in allo control-treated recipients compared with DMOG-treated allo recipients in colon (Figure 4A and B) but not in ileum (Figure 4C). Stronger disruption of colonic epithelial integrity was associated with increased expression of the neutrophil attracting chemokine KC (Figure 3C and 4D) and with increased MPO expression in colon (Figure 4E and F).

FIGURE 4.

FIGURE 4.

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DMOG treatment protects the intestinal epithelium. A, Expression analysis of ZO-1 by IHC in colon sections of allogeneic control and DMOG-treated allogeneic mice at d +7. B, Representative pictures of ZO-1 IHC in colon sections of allogeneic control and DMOG-treated allogeneic mice at d +7. C, Expression analysis of ZO-1 by IHC in ileum sections of allogeneic control and DMOG-treated allogeneic mice at d +7. D, Correlation graphs for KC/CXCL1 with pathology scores in ileum and colon, respectively. E, MPO expression analysis and (F) representative picture in colon of allogeneic control and DMOG-treated allogeneic mice at d +7. G, MPO expression analysis and (H) representative picture in ileum of allogeneic control and DMOG-treated allogeneic mice at d +7. IHC data were analyzed through imageJ software and the Y axis in A, C, E, and G indicates % of positive area for respective antibody. Sample size at d +7 syn control: 3; syn DMOG: 3; allogeneic control: n = 6; DMOG-treated allogeneic: n = 6. Results shown as mean ± SD. Statistical comparisons between groups were made using student’s t test. A P ≤ 0.05 was considered statistically significant. R2 represents coefficient of correlation. DMOG, dimethyl oxalyl glycine; IHC, immunohistochemistry; MPO, myeloperoxidase; ZO-1, zonula occludens-1.

DISCUSSION

In this study, we show that DMOG, a hydroxylase inhibitor, increases survival and ameliorates disease severity in a mouse model of acute GVHD. In the literature, beneficial effects of HIF-stabilizing therapy by PHIs like DMOG have been found for a wide variety of inflammatory diseases such as IBD, colitis, and ischemic disorders.7 Studies have shown that hypoxia occurs concurrently with inflammatory responses,19 lung injury,20 IBD, and colitis in mice.21

Acute GVHD is an immune-mediated disease that results from a complex interaction between immune cells from both the donor and the recipient. Donor T cells are able to recognize minor (or major) HLA disparities between donor and recipient, which are directly related to the development of acute GVHD.22 In acute GVHD, initial donor T-cell expansion occurs early after allogeneic HCT, with T cells being considered the main effector cells mediating acute GVHD. The immunological attack on target recipient organs and tissues by donor allogeneic T cells causes tissue damage in the gastrointestinal tract, liver, lung, and skin.

The initiation of GVHD is necessarily influenced by the cytokine milieu in which it arises, and 3 distinct phases have been described.23,24 We show here that DMOG treatment resulted in a reduction of serum TNF levels and a trend toward reduced levels of IFN-Υ and IL-6. The initial phase of GVHD is triggered by tissue damage and associated loss of mucosal barrier function, primarily in the gastrointestinal tract, which is caused by the conditioning regimens. We observe a reduction in pathologic damage in the gut of DMOG-treated allogeneic mice compared with allogeneic controls as revealed by histological examination at day +7 post HCT. However, no significant difference in pathologic scores is observed in other GVHD target organs early after HCT and not in any organ at day +50. Both MHC I, II and minor Hag mismatches between donor and recipient in this murine model of GVHD result in a rapid early expansion of T cells, which together with conditioning toxicity and a resulting cytokine storm cause early significant mortality.25,26 This initial phase seems more amendable to DMOG treatment mainly by reducing intestinal damage.

One of the key soluble factors in the pathophysiology of acute GVHD is the proinflammatory cytokine TNF-α, which participates in the initiating events that culminate in GVHD as well as amplifies the disease process once established. The importance of TNF-α in this process is supported by a series of experiments demonstrating a correlation between TNF signaling and GVHD.27 TNF-α has both indirect effects, through activation and proliferation pathways of T cells, the main cellular effector of acute GVHD, and direct effects on GVHD target tissues, leading to apoptosis.27,28 TNF-α induced intestinal epithelial cell apoptosis is known to be a major factor for barrier disruption,29 which increases intestinal injury and thereby affecting injury to other GVHD target organs.30

We were unable to detect differences in tissue levels of TNF-α by ELISA in the intestine (data not shown), yet macrophages and T cells, a well-known source of TNF-α in early acute gut GHVD pathophysiology,23,31 responded significantly less to in vitro endotoxin or alloantigen stimulation, respectively (Figure 1I) (T-cell data not shown).

Reduced level of RANTES, MIP-1a, and MIP1b, in the ileum but not in colon of DMOG-treated recipients contribute to the reduced infiltration of T cells in ileum and support prior reports on the roles these chemokines mediating in intestinal GVHD.32,33 Further, in our study, T-cell infiltration in the terminal ileum was reduced after DMOG treatment, while there were no differences in splenic T-cell proliferation or systemic IFN-Υ levels, suggesting that the decreased number of T cells in the gut was rather due to decreased recruitment to target tissue than generally reduced T-cell expansion.

Because TNF-α induces apoptosis via TNF receptor 1, we investigated FADD, one of the critical proteins involved in this pathway.34 FADD has a pivotal role in TNF-mediated apoptosis and serves as one of the potential targets of DMOG. We observed significantly reduced FADD expression in terminal ileal sections in allogeneic recipients treated with DMOG. This observation is associated with reduced organ injury mainly in the gut in the DMOG-treated allogeneic transplant group at early stage, alongside reduced serum proinflammatory cytokines and improved survival. Hindryckx et al17 found that HIF-1α binds effectively to the endogenous FADD promoter and has a potential role in transcriptional repression of FADD. We confirm here the protective effect of DMOG on TNF-α induced intestinal epithelial cell apoptosis, mediated by DMOG stabilized HIF-1α interaction with FADD. Hindryckx et al17 have previously shown that DMOG treatment in their model resulted in a stable increase of functionally active intestinal epithelial HIF-1α in the terminal ileum as well as in vitro.

LPS stimulation evokes classical (M1) activation of macrophages35 and activation of the alternative (M2) macrophage phenotype is associated with LPS tolerance.36 It was previously shown that DMOG attenuates LPS induced NF-κB signaling in vivo and induces tolerance to subsequent LPS challenge by promoting M2 polarization in macrophages within the peritoneal cavity, resulting in the downregulation of proinflammatory cytokines such as TNF-α.37 Hill et al23 demonstrated an important role for TNF production by intestinal macrophages in response to TBI conditioning, driving GVHD development. Furthermore, earlier studies from our group showed that about half of serum TNF on day 7 after murine allo-HCT is T cell–derived, the other half macrophage–derived, and that donor CD4+ T cell–derived TNF significantly contributes to GVHD and transplant-related mortality.31 In this study, we did not see differences with DMOG treatment in splenic T-cell expansion nor did we find major changes in serum IFN-Υ levels, a cytokine classically secreted by alloreactive T cells. However, TNF levels were significantly reduced both in serum of DMOG-treated allogeneic recipients and in supernatants of LPS stimulated, DMOG-treated peritoneal macrophages, suggesting an alternative cellular source of TNF, for example, macrophages being affected by DMOG, contributing to the observed reduction in GVHD and improvement in survival.

It has been shown that DMOG treatment maintains the intestinal barrier.18 We measured epithelial integrity by measuring the expression of ZO-1 in ileum and colon. Significant loss of ZO-1 was observed in colon but not in ileum, suggesting worse disruption of the epithelial barrier in the large intestine.38 DMOG treatment preserved ZO-1 expression and decreased KC and MPO expression in the large bowel. This supports the role of bacterial translocation across the damaged epithelium for neutrophil recruitment to the intestinal tract, which enhances graft versus host disease by promoting further tissue damage.39

The contribution of neutrophils to GVHD severity is mediated by the production of large amounts of reactive oxygen species (ROS),39 and various ROS are associated with a disruption of tight junctions and adherens junctions.40 We did not observe significant loss of ZO-1 expression in the small bowel despite increased T-cell infiltration and elevated tissue expression of proinflammatory markers. Similarly, no changes in ZO-1 expression were described for small intestinal biopsies by Nalle et al41 despite increased MLCK210 mediated epithelial tight junction permeability along with increased T-cell infiltration in the jejunum during GVHD. The differences in severity of epithelial disruption between small and large bowel potentially relate to differences in the microbiome or could be the result of kinetic differences of damage evolution warranting further investigation.

In conclusion, DMOG treatment after allo-HCT results in decreased intestinal GVHD in a well-established murine model of acute GVHD by modulating intestinal injury and epithelial protection. Currently, several orally active PHIs inhibitors are already in late phase clinical trials.42 Roxadustat is an oral HIF PHI and recently found to be effective for the treatment of anemia in chronic kidney disease with an established safety profile.43 It was recently shown, that DMOG synergizes with cyclosporine in a model of experimental T cell–mediated colitis,18 therefore, given the barrier protective effects of PHIs on the gut epithelium, combination therapy of PHI with standard GVHD prophylaxis may beneficial and warrants further investigation.

Acknowledgments

This work has been funded by Louisiana State University Health Sciences Center, Shreveport, Louisiana and University of Kentucky, Lexington, Kentucky, USA.

This research was supported by the Biospecimen Procurement & Translational Pathology Shared Resource Facility of the University of Kentucky Markey Cancer Center (P30CA177558). UK Flow Cytometry and Cell Sorting Core Facility was supported by the Office of the Vice President for Research, the Markey Cancer Center and an NCI Center Core Support Grant (P30CA177558). UK COBRE pathologic Core, University of Kentucky, National Institutes of General Medical Sciences, NIH grant 8 P20 GM103527.

Footnotes

G.C.H. and S.P have received project-unrelated research funding from Takeda, Pharmacyclics, and JAZZ Pharmaceuticals. G.C.H. has advisory roles in the field of GVHD with Incyte and JAZZ Pharmaceuticals. The other authors declare no conflicts of interest.

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