Novel Treatment of Chronic Graft-Versus-Host Disease in Mice Using the ER Stress Reducer 4-Phenylbutyric Acid

Mukai, Shin; Ogawa, Yoko; Urano, Fumihiko; Kudo-Saito, Chie; Kawakami, Yutaka; Tsubota, Kazuo

doi:10.1038/srep41939

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Published: 06 February 2017

Novel Treatment of Chronic Graft-Versus-Host Disease in Mice Using the ER Stress Reducer 4-Phenylbutyric Acid

Shin Mukai^1,2,
Yoko Ogawa¹,
Fumihiko Urano³,
Chie Kudo-Saito²,
Yutaka Kawakami² &
…
Kazuo Tsubota¹

Scientific Reports volume 7, Article number: 41939 (2017) Cite this article

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Abstract

Chronic graft-versus-host disease (cGVHD) is a notorious complication of allogeneic hematopoietic stem cell transplantation and causes disabling systemic inflammation and fibrosis. In this novel study, we focused on a relationship between endoplasmic reticulum (ER) stress and cGVHD, and aimed to create effective treatment of cGVHD. A series of experiments were conducted using a mouse model of cGVHD. Our data suggested (1) that ER stress was elevated in organs affected by cGVHD and (2) that 4-phenylbutyric acid (PBA) could reduce cGVHD-induced ER stress and thereby alleviate systemic inflammation and fibrosis. Because fibroblasts are thought to be implicated in cGVHD-elicited fibrosis and because macrophages are reported to play a role in the development of cGVHD, we investigated cGVHD-triggered ER stress in fibroblasts and macrophages. Our investigation demonstrated (1) that indicators for ER stress and activation markers for fibroblasts were elevated in cGVHD-affected lacrimal gland fibroblasts and (2) that they could be reduced by PBA. Our work also indicated that splenic macrophages from PBA-dosed mice exhibited the lower levels of ER stress and M2 macrophage markers than those from cGVHD-affected mice. Collectively, this study suggests that the reduction of ER stress utilizing PBA can be a clinically translatable method to treat systemic cGVHD.

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Introduction

Chronic graft-versus-host disease (cGVHD) is a disabling complication after allogeneic hematopoietic stem cell transplantation (HSCT) and has a highly negative impact on patients’ quality of life. Unfortunately, there has been painfully slow progress in developing effective treatment of cGVHD thus far. Acute GVHD (aGVHD) has been studied intensively, and there is a growing understanding of aGVHD. In contrast, sparse information about mechanisms of cGVHD has been provided. Although immunosuppressant such as cyclosporine has been utilized to cure cGVHD in medical practice, it has been only partially effective for cGVHD. Most recently, several strategies to treat cGVHD have been reported^1,2,3,4. However, there is still vast scope for improvement in the treatment of cGVHD. aGVHD is initiated by preparative regimens which induce host tissue damage, proinflammatory cytokine release and chemokine production^5,6,7,8. Donor T-cells subsequently recognize host alloantigens as foreign bodies and thereby attack recipient cells^9,10. The two types of GVHD should be classified by clinical manifestations, not by time after HSCT¹¹. However, aGVHD typically appears within 100 days after HSCT, and cGVHD usually manifests 6 months or later after HSCT and resembles autoimmune diseases^12,13. While cGVHD is largely unexplored, it has been reported that cellular senescence is detrimentally involved in ocular cGVHD and that cGVHD can be regarded as an age-associated disease¹⁴. In clinical settings, gray eyebrows, skin wrinkles and conjunctival cancer are observed, which suggests that recipient cells as well as donor immune cells are senescent in cGVHD^14,15.

Literature precedent indicates that endoplasmic reticulum (ER) stress plays a contributory role in chronic inflammation and age-related diseases^16,17. The ER is a cellular organelle essential for cells to function properly. When proteins are synthesized in the ER, they need to be folded adequately and ER chaperones assist the protein folding¹⁸. However, hypoxia, calcium ion depletion, oxidative injury, viral infections and inflammatory cytokines prevent the ER from performing its normal protein folding^19,20. When unfolded and misfolded proteins are accumulated in the ER, it causes ER stress and the following three transmembrane proteins are consequently activated to initiate the unfolded protein response (UPR): inositol requiring (IRE) 1α, PKR-like ER kinase (PERK) and activating transcription factor (ATF) 6α¹⁸. However, if the UPR is prolonged or unsuccessful, inflammatory and apoptotic pathways are activated¹⁸. Hence, the unsuccessful UPR leads to the expression of (1) the proinflammatory molecules thioredoxin interaction protein (TXNIP) and transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and (2) the apoptotic protein C/EBP homologous protein (CHOP)^{21,22,23,24,25,26}.

In this study, we aimed to devise effective treatment of cGVHD. Based on the previous findings, we envisaged (1) that ER stress would be increased in cGVHD-impaired organs and (2) that mitigation of ER stress could be effective treatment of cGVHD. To achieve this goal, we used 4-phenylbutyric acid (PBA) as an ER stress reducer²⁷. (Supplementary Figure 1A) PBA is commercially available and can be an inexpensive starting material in organic synthesis²⁸. In clinical settings, PBA is approved by FDA and has been utilized to cure urea cycle disorders²⁷. Furthermore, several articles demonstrate that PBA can be effective for chronic inflammation and age-related diseases such as type 2 diabetes and obesity-caused chronic inflammation through mitigation of ER stress^29,30. Hence, it was envisioned that PBA could be a safe and efficacious drug for cGVHD, in which systemic inflammation and fibrosis are highly problematic. Herein, we report the world-first strategy to treat cGVHD using the ER stress reducer PBA.

Results

Elevation of ER stress markers in cGVHD-affected organs

The initial venture toward our overarching aim was to investigate whether ER stress was elevated in cGVHD-affected organs by measuring ER stress markers. Real-time quantitative PCR (qPCR) and immunoblot analysis suggested (1) that the ER stress indicators GRP78, CHOP, p-PERK, p-eIF2α, and p-IRE1α were increased in murine organs affected by cGVHD compared with control subjects and (2) that accordingly the following two inflammatory molecules were activated and/or augmented in the cGVHD-impaired organs: NF-κB and TXNIP (Fig. 1A–C). As evidenced by electron micrographs, in cGVHD-impaired lacrimal gland epithelia, accumulated misfolded/unfolded proteins caused the ER to expand (Fig. 1D). Conversely, the ER in the controls seemed intact (Fig. 1D). These observations are indicative of the elevation of ER stress in cGVHD-affected organs.

**Figure 1: ER stress markers in cGVHD target organs.**

Deactivation of the ER stress-induced inflammatory pathways utilizing PBA

Once we found that ER stress was increased in the organs impaired by cGVHD, our next attempt was to treat cGVHD through reduction of ER stress by the use of PBA. As depicted in the Methods section, we administered PBA or the solvent-vehicle to mice which underwent allogeneic BMT. Immunoblot analysis indicated that organs collected from the PBA-treated mice exhibited the lower protein levels of GRP78, CHOP, p-PERK, p-eIF2α, and p-IRE1αcompared with those from the vehicle-treated mice (Fig. 2A,B; Supplementary Figure 2A,B). Consequently, the associated proinflammatory molecules NF-κB and TXNIP were subdued in the organs treated with PBA compared to their vehicle-treated counterparts (Fig. 2A,B; Supplementary Figure 2A,B).

**Figure 2: Suppression of cGVHD-caused ER stress by PBA.**

Histological observations of cGVHD target organs

HE and Mallory pictures of cGVHD-susceptible organs indicate that the reduction of ER stress using PBA can be efficacious treatment of cGVHD (Fig. 3). As judged by the HE pictures, inflammatory cell infiltration was repressed in the PBA-medicated organs compared with their vehicle-medicated counterparts (Fig. 3A; Supplementary Figure 3A). Particularly, (1) the intestinal villi of the PBA-treated small and large intestines seemed intact in contrast to those treated with the solvent-vehicle, (2) the vehicle-treated skin was thickened, lost the fatty tissues and had the elevated density of collagen bundles by contrast with the PBA-treated skin, (3) meibomian glands in the vehicle-medicated eyes were decreased and shrunk compared with their PBA-medicated counterparts and (4) the thinning and damage in vehicle-injected conjunctival epithelia were observed in contrast to their PBA-injected counterparts, which suggested that a symblepharon could be prevented by systemic injection of PBA (Fig. 3A, Supplementary Figure 3A). As indicated by the immunostaining and subsequent counting of CD45⁺ cells, the number of inflammatory cells in the PBA-treated organs was considerably lower than that in their vehicle-treated counterparts (Fig. 3B,E; Supplementary Figure 3B,D). These observations are also indicative of the suppression of immune cell migration and proliferation in the PBA-treated organs. Furthermore, systemic fibrosis is one of the most serious problems in cGVHD³¹. Mallory’s staining revealed that cGVHD-induced fibrosis was substantially subdued in the PBA-medicated organs compared with the vehicle-medicated ones (Fig. 3C; Supplementary Figure 3C). Electron micrographs of the lacrimal glands indicate that in the case where the lacrimal glands were treated with the solvent-vehicle, (1) the ER of the epithelial and endothelial cells were expanded due to the accumulation of unfolded/misfolded proteins, (2) there was a large amount of cell debris in the stroma and (3) mitochondria in the epithelial and endothelial cells were damaged (Fig. 3D). Electron micrographic analysis of the small intestine also demonstrated that when the small intestine was treated with the solvent-vehicle, its microvilli were demolished and the neighboring tissues were damaged (Fig. 3D). These histological findings were not observed in the PBA-treated lacrimal glands or small intestine. In addition, PAS staining revealed (1) that goblet cells in the PBA-injected small intestine and conjunctival epithelia outnumbered those in their vehicle-injected counterparts and (2) that the structures of intestinal and conjunctival mucosal membranes were retained by systemic injection of PBA (Supplementary Figure 4A,B). As judged by all the histological observations, PBA can alleviate systemic inflammation and fibrosis elicited by cGVHD-related ER stress.

**Figure 3: Mitigation of cGVHD-elicited systemic inflammation and fibrosis using the ER stress mitigator PBA.**

Reduction of inflammatory and pro-fibrotic mediators by the use of PBA

To examine more closely whether PBA could mitigate cGVHD-caused systemic inflammation, ELISA was conducted to measure the levels of the inflammatory molecules MCP-1, TNF-α and IFN-γ in sera collected from the PBA- and vehicle-injected mice. The sera from the PBA-medicated mice exhibited the lower protein levels of MCP-1, TNF-α and IFN-γ in comparison to those from their vehicle-medicated counterparts. (Figure 4A) Moreover, we performed more meticulous investigation into the alleviation of cGVHD-triggered systemic fibrosis, and immunoblot analysis revealed that the pro-fibrotic mediator connective tissue growth factor (CTGF) was overexpressed in the vehicle-treated organs, yet it was not observed in those treated with PBA (Fig. 4B,C; Supplementary Figure 5A,B). These data support that PBA can repress cGVHD-induced ER stress and consequently mitigate systemic inflammation and fibrosis.

**Figure 4: Reduction of indicators for inflammation and fibrosis utilizing the ER stress alleviator PBA.**

Correlation between ER stress and dysfunctional fibroblasts

Systemic fibrosis arising from cGVHD is a serious problem and causes multiple organ failure³¹. Although the mechanisms of cGVHD-triggered fibrosis still need to be elucidated, fibroblasts are conceivably implicated in the development of fibrosis³². Several biological signals stimulate fibroblasts so that they can play an integral role in several biological processes such as wound healing^33,34. However, if fibroblasts are activated in an uncontrolled manner, they produce abnormal collagen bundles and thereby induce severe fibrosis although there is little information about what causes their abnormality^33,34. Hence, we strived to unravel a relationship between cGVHD-elicited ER stress and dysfunctional fibroblasts. To accomplish this goal, we cultured fibroblasts from the PBA- and vehicle-treated murine lacrimal glands as described in the Methods section³⁵. As confirmed by immunoblot assays, the cultured lacrimal gland fibroblasts (expressing α-SMA) were not contaminated with epithelial cells (cytokeratin positive cells) (Supplementary Figure 13), and the following experiments were carried out by the use of sufficiently pure fibroblasts. Immunoblot analysis of the cultured fibroblasts suggested that GRP78, CHOP, phosphorylated PERK, phosphorylated eIF2α and phosphorylated IRE1α were greatly suppressed by treatment of the mice with PBA in comparison to the solvent-vehicle (Fig. 5A). Accordingly, (1) the protein levels of HSP47 and CTGF, markers for fibroblast activation and fibrosis, respectively, (2) the production of MCP-1 and (3) the mRNA level of IL-6 were reduced in the PBA-treated fibroblasts compared with their vehicle-treated counterparts (Fig. 5A–D).

**Figure 5: Alleviation of cGVHD-triggered ER stress in lacrimal gland fibroblasts utilizing the ER stress attenuator PBA.**

Link between ER stress and the polarization of macrophages

Macrophages serve a crucial role in immune responses and inflammation, and an earlier article indicates that senescent macrophages are detrimentally linked with the pathogenic process of cGVHD¹⁴. Hence, we scrutinized a relationship between cGVHD-induced ER stress and malfunctioning macrophages. In the first instance, immunostaining was conducted to investigate whether macrophages in the murine lacrimal glands expressed the ER stress indicator CHOP. As judged by fluorescence images of the lacrimal glands, when mice were medicated with the solvent-vehicle, macrophages expressing CHOP migrated to the tissues (Fig. 6A). In contrast, no such macrophages were observed in the lacrimal glands collected from the PBA-medicated mice (Fig. 6A). Our subsequent endeavors were to gain greater insights into ER stress in cGVHD-affected macrophages. Toward this end, we cultured splenic macrophages from PBA- and vehicle-injected mice as depicted in the Methods section³⁶. As evidenced by immunoblot analysis, the induced macrophages (expressing CD68) were not adulterated with T-cells (CD3⁺ cells) or B-cells (CD19⁺ and/or CD20⁺ cells) (Supplementary Figure 14), and the following results were gleaned utilizing highly pure macrophages. Immunohistochemistry revealed that the vehicle-medicated splenic macrophages expressed CHOP in contrast to the PBA-medicated ones (Fig. 6B). Immunoblot analysis of the macrophages also indicated that in the case where they were treated with PBA, the ER stress markers GRP78, CHOP, phosphorylated PERK, phosphorylated eIF2α and phosphorylated IRE1α were substantially repressed compared with the solvent-vehicle (Fig. 6C,D). We then conducted qPCR to examine the gene expression of (1) the M1 macrophage markers IL-1β, IL-6 and MCP-1 and (2) the M2 macrophage markers TGF-β and IL-10 in the splenic macrophages³⁷. As demonstrated by the data, the mRNA expression of TGF-β and IL-10 was increased in the vehicle-treated macrophages in comparison to the PBA-treated ones (Fig. 6E). Conversely, when macrophages were treated with PBA, they showed the higher gene expression of IL-1β and IL-6 than those treated with the solvent-vehicle (Fig. 6E). These outcomes are indicative of the connection between cGVHD-elicited ER stress in macrophages and their differentiation into an alternatively activated phenotype.

**Figure 6: Mitigation of cGVHD-caused ER stress in macrophages by the use of PBA.**

Discussion

cGVHD is a major complication following HSCT and can be deadly for some patients. Although cGVHD has been studied intensively over the last decades, sluggish progress in developing efficacious treatment of cGVHD has been made. Patients with cGVHD have been treated with immunosuppressant such as cyclosporine in many cases, however the desired results have been elusive. Hence, clinical settings are in desperate need of effective methods to cure cGVHD. A survey of literature suggests (1) that cellular senescence in donor immune cells and recipient cells detrimentally contributes to the pathogenic process of cGVHD¹⁴ and (2) that ER stress serves a detrimental role in the development of chronic inflammation and age-associated diseases^29,30. These reports have led us to a new hypothesis (1) that cGVHD-affected organs show the elevated level of ER stress and (2) that alleviation of ER stress can be a robust strategy to ease severe symptoms caused by systemic cGVHD (Fig. 7).

**Figure 7: Conceivable association between cGVHD and ER stress.**

In this proof-of-concept research, cGVHD was experimentally induced in mice. We investigated the pathogenesis of chronic GVHD in the human lacrimal glands from 2001 to 2009^{32,38,39,40,41}. Our investigation demonstrated that the pathogenesis of lacrimal gland cGVHD in this murine model was reminiscent of that in patients with chronic GVHD^1,14,31,42. Excessive inflammation and fibrosis observed in this mouse model greatly resemble those seen in human samples. Furthermore, our previous reports showed that systemic inflammation and fibrosis were observed in this mouse model^1,31. Hence, we chose this animal model to tackle systemic cGVHD.

Toward the ultimate goal, we initially elucidated (1) the elevation of ER stress and (2) the increased activity and/or expression of its corresponding proinflammatory molecules such as NF-κB and TXNIP in cGVHD-affected organs. Drobyski and co-workers have demonstrated that NF-κB detrimentally correlates with the pathogenic process of aGVHD and that inhibition of NF-κB can alleviate aGVHD⁵. Our study suggests that NF-κB is activated in cGVHD-affected organs and that it results partly from unresolved ER stress caused by cGVHD.

With respect to the involvement of TXNIP in the pathogenesis of cGVHD, there is no literature precedent which mentioned an association between TXNIP and cGVHD. Our survey of previous articles has revealed (1) that TXNIP is generated in the downstream of the PERK and IRE1α pathways, (2) that it serves a pivotal role in a crossroad where cells subjected to ER stress determine whether to restore homeostasis or undergo apoptosis and (3) that it induces extensive inflammation by activating the inflammasome nod-like receptor family, pyrin domain containing 3 (NLRP3)^22,23,24. Thus, our investigation indicates that the ER stress-elicited expression of TXNIP can correlate with disabling symptoms of cGVHD.

We obtained these scientifically and medically important outcomes, and our subsequent endeavors were to develop effective therapeutics to treat cGVHD through mitigation of ER stress. We initially attempted to reduce cGVHD-induced ER stress utilizing the ER stress mitigator rapamycin^4,43,44,45 (Supplementary Figure 1B) and found that it could ease ER stress and be efficacious for systemic cGVHD (data not shown). However, it also acts as an immunosuppressive drug and has been reported to induce extensive side-effects such as viral and fungal infections in clinical settings^46,47. These reports prompted us to circumvent the setbacks and search for a safer compound to mitigate ER stress, and we chose 4-phenylbutyric acid (PBA), which is a commercially available aromatic fatty acid and can be used as an affordable starting material in synthetic organic chemistry^48,49. In medical practice, PBA has been proven to be sufficiently safe to treat urea cycle disorders²⁷. Literature precedent suggests that PBA can attenuate ER stress, since it serves as a chemical chaperone and assists the ER in dealing with unfolded/misfolded proteins⁵⁰. As demonstrated by previous reports, PBA can be effective for type 2 diabetes and obesity-elicited chronic inflammation in adipose tissues by lowering ER stress^29,30. Taken together, it was envisioned that PBA could be a safe and powerful tool to reduce cGVHD-caused ER stress.

A series of our experiments demonstrated that cGVHD-caused ER stress in murine organs could be mitigated by PBA and that accordingly the ER stress-associated proinflammatory molecules NF-κB and TXNIP were greatly subdued.

With these encouraging results, we subsequently investigate the state of cGVHD-vulnerable organs. Histological, immunohistochemical and electron microscopic analysis of these tissues was conducted. The HE pictures and immunofluorescence images of the cGVHD target organs demonstrated that the PBA-medicated tissues were less inflamed than the vehicle-medicated ones. Our survey of previous reports shows (1) that PBA can alleviate inflammation by reducing ER stress in endothelial cells and (2) that when ER stress is elevated, mitochondria are damaged^51,52,53. As demonstrated by electron micrographs of the lacrimal glands, when mice were treated with the solvent-vehicle, mitochondria in the blood vessels were impaired. In contrast, this phenomenon was not observed in the lacrimal glands collected from the PBA-medicated mice. Thus, these findings indicate that PBA can protect blood vessels from cGVHD-caused ER stress and thereby suppress immune cell migration to tissues.

Mallory staining and immunoblot analysis of the fibrosis marker CTGF demonstrate that systemic fibrosis was subdued by PBA. Our literature search has revealed that epithelial to mesenchymal transition (EMT) correlates with fibrosis observed in human ocular cGVHD and that TXNIP is linked to EMT^54,55. Hence, PBA is conceivably able to attenuate cGVHD-triggered systemic fibrosis, partly because it precludes EMT by lowering the expression of TXNIP. Furthermore, as indicated by ELISA, sera collected from the PBA-dosed mice displayed the lower levels of the inflammatory molecules MCP-1, TNF-α and IFN-γ in comparison to those obtained from their vehicle-dosed equivalents. CTGF is known to serve as an indicator for fibrosis⁵⁶, and our immunoblot analysis of the cGVHD-susceptible organs indicates that PBA can prevent the cGVHD-elicited overexpression of CTGF. These results have underpinned our claim that reduction of ER stress can attenuate disabling symptoms caused by systemic cGVHD, and have encouraged us to conduct more painstaking investigation into the correlation between ER stress and the pathogenic process of cGVHD. As mentioned above, cGVHD causes extensive fibrosis in various organs. Although there is sparse information about the mechanisms of cGVHD-induced fibrosis, a previous report demonstrates that the murine lacrimal glands impaired by cGVHD have the increased number of activated fibroblasts and that they are associated with the progression of fibrosis³². Hence, we focused on fibroblasts in the lacrimal glands and scrutinized a correlation between cGVHD-induced ER stress and disordered fibroblasts. Our data suggest (1) that cGVHD-caused ER stress activates fibroblasts in a detrimental way and thereby triggers extensive fibrosis and (2) that PBA can prevent fibroblasts from dysfunctioning through alleviation of ER stress.

Once we gained novel insights into how ER stress affected the function of fibroblasts, our focus was turned to macrophages. A previous study demonstrates that senescent macrophages serve a causative role in the development of ocular cGVHD¹⁴. Other workers have shown (1) that ER stress promotes the polarization into M2 macrophages and (2) that M2 macrophages are implicated in fibrosis-related diseases^57,58,59. These reports captured our interest and motivated us to scrutinize the relationship between cGVHD-induced ER stress and M2 macrophage differentiation. Judging from immunofluorescence images, the cGVHD-impaired murine lacrimal glands had macrophages expressing the ER stress marker CHOP, whereas it was not the case with the PBA-medicated murine lacrimal glands. These observations indicate that macrophages subjected to ER stress are linked to the progression of cGVHD. Furthermore, splenic macrophages from PBA- and vehicle-medicated mice were induced and subsequently analysed utilizing sophisticated methods. Both immunohistochemistry and immunoblot analysis demonstrated that the PBA-treated splenic macrophages showed the lower protein levels of ER stress markers compared to their vehicle-treated counterparts. Quantitative mRNA analysis suggests (1) that the mRNA levels of the M2 markers TGF-β and IL-10 are enhanced in the splenic macrophages treated with the solvent-vehicle and (2) that the gene expression of the M1 markers IL-1β and IL-6 is increased in those treated with PBA. Hence, it is conceivable (1) that cGVHD-induced ER stress promotes macrophages to differentiate into an alternatively activated phenotype and (2) that PBA can mitigate cGVHD-induced fibrosis by reducing ER stress in macrophages and transforming them into a classically activated phenotype.

Collectively, considering the safety and affordability of the ER stress reducer PBA, the reduction of cGVHD-caused ER stress can be an innovative and robust strategy for the treatment of systemic cGVHD and open up new prospects to cure other immune-mediated diseases associated with ER stress.

Methods

Eight-week-old B10.D2 and BALB/c mice were purchased from Sankyo Laboratory, Inc. (Tokyo, Japan). All the scientific experiments on mice were performed according to the Animal Welfare Act at Keio University School of Medicine. Our protocols for experiments on animals were approved by the animal care and use committee at Keio University (Approval Number: 09152). We also complied with the ARRIVE guidelines⁶⁰.

Bone marrow transplantation

Bone marrow transplantation (BMT) was conducted to furnish a murine model of cGVHD⁴². In the case where the donors were B10.D2 mice and the recipients were BALB/c mice, it was allogeneic BMT to produce a murine model of cGVHD. In contrast, BMT from BALB/c to BALB/c mice was syngeneic BMT, and therefore cGVHD did not occur in the transplant recipients. The recipient mice without cGVHD served as syngeneic control subjects. The recipients were irradiated with 700cGy prior to the BMT, and the lethal irradiation was preformed using a Gammacel 137 Cs source (Hitachi Medico, Ltd, Tokyo, Japan). A suspension containing 1 × 10⁶ bone marrow cells and 2 × 10⁶ spleen cells from the donors was administered to each of the recipient mice via tail vein. The donor cells were suspended in RPMI 1640 (Life Technologies Japan Ltd, Tokyo, Japan).

Treatment of allogeneic BMT recipient mice with PBA

We performed BMT as described in the Methods section, and the allogeneic BMT recipient mice were divided into 2 groups. One group was treated with PBA (10 mg/kg) (Aldrich, St. Louis, MO), and the other was given the solvent-vehicle PBS (pH 7.4) by intraperitoneal injection. We administered PBA or the solvent-vehicle to the allogeneic BMT recipients once per day from Day 10 to Day 27 after BMT. They were sacrificed Day 28 after BMT. In this study, the following cGVHD-susceptible organs were analyzed: extra-orbital lacrimal glands, the proximal part of small intestine, dorsum skin, liver, salivary glands, lung, large intestine and eyes.

Culture of fibroblasts from murine lacrimal glands

Fibroblasts derived from the murine lacrimal glands were cultured using the well-established method reported by Yaguchi et al.³⁵. The murine lacrimal glands were collected and subsequently cut into small pieces. The tissue fragments were incubated at 37 °C in DMEM (Life Technologies Japan Ltd, Tokyo, Japan) containing 5% of fetal bovine serum (FBS) (Sigma, St. Louis, MO) and 5% of antibiotics. The antibiotics were a 1:1 aqueous mixture of streptomycin sulfate (Meiji Seika Pharma Co., Ltd, Tokyo, Japan) and benzylpenicillin potassium (Meiji Seika). Fibroblasts started growing from the minced pieces after 3–4 days. The fibroblasts were cultured in DMEM with 5% of FBS and 5% of antibiotics, and used for experiments after 3–5 passages. Trypsin (Becton Dickinson, Franklin Lakes, NJ) was used to detach the fibroblasts from the culture dishes.

Culture of splenic macrophages

We cultured murine splenic macrophages according to the method of Alatery and Besta with slight modifications³⁶. Splenocytes suspended in RPMI with 5% of FBS were seeded in culture dishes and left at 37 °C overnight. The floating cells were then removed. The desired cells attached to the dishes were cultured in RPMI containing recombinant murine M-CSF (5 ng/mL) (Peprotech, Rocky Hill, NJ), 5% of FBS and 5% of antibiotics for 7 days. The induced macrophages were subsequently detached from the culture dishes utilizing accutase (Thermo Fisher Scientific, Waltham, MA) and used for experiments.

Immunostaining of cultured macrophages

The induced macrophages were placed in 8-well chamber slides (Fibronectin Culture Slide; Corning Inc., Corning, NY) and fixed with 10% neutral buffered formlain. They were then blocked with methanol containing 0.3% hydrogen peroxide at RT for 30 min and incubated with the primary antibodies CD68 (AbD Serotec) and CHOP (Santa Cruz) at 4 °C overnight. The sections were subsequently treated with goat anti-mouse IgG (H + L) secondary antibody, Alexa Fluor 568 conjugate (Molecular Probes, Eugene, OR) and 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies) at RT for 45 minutes and mounted with an anti-fading mounting medium (Dako). Fluorescence images were taken with an LSM confocal microscope (Carl Zeiss).

Quantitative polymerase chain reaction

Total RNA was extracted from extra-orbital lacrimal glands, the proximal part of small intestine, dorsum skin, liver, and cultured fibroblasts and macrophages using an miRNeasy mini kit (Qiagen, Valencia, CA), and the corresponding complementary DNA was synthesized utilizing a Rever Tra Ace qPCR RT Kit (Toyobo Co. Ltd. Osaka, Japan). The primers required to analyze the mRNA expression of the following genes by the use of TaqMan real-time polymerase chain reaction (PCR) were purchased from Applied Biosystems (Applied Systems Inc, Streetsville, Ontario, Canada): the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glucose regulated protein 78 (GRP78), Interleukin-1β (IL-1β), IL-6, IL-10, macrophage chemoattractant protein-1 (MCP-1) and transcription growth factor-β (TGF-β). Quantitative real-time PCR was performed using the Step One Plus system (Applied Biosystems). The 2^−ΔΔCT method was used to analyze the data obtained, and GAPDH was used as an internal standard to measure the expression of mRNA.

Statistical analysis

Statistical significance was determined by the use of unpaired Mann–Whitney U test. Differences are considered significant in the case of P < 0.05. The acquired data are presented as means ± SD.

Additional Information

How to cite this article: Mukai, S. et al. Novel Treatment of Chronic Graft-Versus-Host Disease in Mice Using the ER Stress Reducer 4-Phenylbutyric Acid. Sci. Rep. 7, 41939; doi: 10.1038/srep41939 (2017).

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References

Yaguchi, S. et al. Angiotensin II type 1 receptor antagonist attenuates lacrimal gland, lung, and liver fibrosis in a murine model of chronic graft-versus-host disease. PLoS One 8, e64724 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Kim, S. W. et al. Effect of roflumilast, novel phosphodiesterase-4 inhibitor, on lung chronic graft-versus host disease in mice. Exp. Hematol. 44, 332–341 (2016).
Article CAS PubMed Google Scholar
Yoon, H. K., Lim, J. Y., Kim, T. J., Cho, S. C. & Min, C. K. Effects of pravastatin on murine chronic graft-versus-host disease. Transplantation 90, 853–860 (2010).
Article CAS PubMed Google Scholar
Ai, D. et al. Activation of ER stress and mTORC1 suppresses hepatic sortilin-1 levels in obese mice. J. Clin. Invest. 122, 1677–1688 (2012).
Article CAS PubMed PubMed Central Google Scholar
Vodanovic-Jankovic, S., Hari, P., Jacobs, P., Komorowski, R. & Drobyski, W. R. NF-kB as a target for the prevention of graft-versus-host disease: comparative efficacy of bortezomib and PS-1145. Blood 107, 827–834 (2006).
Article CAS PubMed PubMed Central Google Scholar
Xun, C. Q., Thompson, J. S., Jennings, C. D., Jennings, S. A. & Widmer, M. B. Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute and chronic graft-versus-host in H-2 incompatible transplanted SCID mice. Blood 83, 2360–2367 (1994).
Article CAS PubMed Google Scholar
Gonzalez, M. et al. The balance between donor T cell anergy and suppression versus lethal graft-versus-host disease is determined by host conditioning. J. Immunol. 169, 5581–5589 (2002).
Article CAS PubMed Google Scholar
Reddy, P. Pathophysiology of acute graft-versushost disease. Hematol. Oncol. 21, 149–161 (2003).
Article PubMed Google Scholar
Shlomchik, W. D. et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 285, 412–415 (1999).
Article CAS PubMed Google Scholar
Zhang, Y. et al. APCs in the liver and spleen recruit activated allogeneic CD8+ T cells to elicit hepatic graft-versus-host disease. J. Immunol. 169, 7111–7118 (2002).
Article CAS PubMed Google Scholar
Vigorito, A. C. et al. Evaluation of NIH consensus criteria for classification of late acute and chronic GVHD. Blood 114, 702–708 (2009).
Article CAS PubMed PubMed Central Google Scholar
Toubai, T., Sun, Y. & Reddy, P. GVHD pathophysiology: is acute different from chronic? Best Pract. Res. Clin. Haematol. 21, 101–117 (2008).
Article CAS PubMed Google Scholar
Ceredig, R. Graft-versus-host disease: who’s responsible? Immunol. Cell. Biol. 90, 253–254 (2012).
Article CAS PubMed Google Scholar
Kawai, M. et al. Expression and localization of aging markers in lacrimal gland of chronic graft-versus-host disease. Sci. Rep 3, 2455 (2013).
Article PubMed PubMed Central Google Scholar
Mohty, M. & Apperley, J. F. Long-term physiological side effects after allogeneic bone marrow transplantation. Hematology Am Soc Hematol Educ Program 229–236 (2010).
Brown, M. K. & Naidoo, N . The endoplasmic reticulum stress response in aging and age-related diseases. Front. Physiol. 3, 263 (2012).
PubMed PubMed Central Google Scholar
Garg, A. D., Kaczmarek, A., Krysko, O., Vandenabeele, P., Krysko, D. V. & Agostinis, P. ER stress-induced inflammation: does it aid or impede disease progression? Trends Mol. Med. 18, 589–598 (2012).
Article CAS PubMed Google Scholar
Lee, A. S. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35, 373–381 (2005).
Article CAS PubMed Google Scholar
Brozzi, F. et al. Cytokines induce endoplasmic reticulum stress in human, rat and mouse beta cells via different mechanisms. Diabetologia 10, 2307–2316 (2015).
Article CAS Google Scholar
Bhandary, B., Marahatta, A., Kim, H. R. & Chae, H. J. An involvement of oxidative stress in endoplasmic reticulum stress and its associated diseases. Int. J. Mol. Sci. 14, 434–456 (2013).
Article CAS Google Scholar
Tam, A. B., Mercado, E. L., Hoffmann, A. & Niwa, M. ER stress activates NF-κB by integrating functions of basal IKK activity, IRE1 and PERK. PLoS One 7, e45078 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Anthony, T. G. & Wek, R. C. TXNIP switches tracks toward a terminal UPR. Cell Metab. 16, 135–137 (2012).
Article CAS PubMed Google Scholar
Lerner, A. G. et al. IRE1alpha induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress. Cell Metab. 16, 250–264 (2012).
Article CAS PubMed PubMed Central Google Scholar
Oslowski, C. M. et al. Thioredoxin-interacting protein mediates ER stress-induced beta cell death through initiation of the inflammasome. Cell Metab. 16, 265–273 (2012).
Article CAS PubMed PubMed Central Google Scholar
Oyadomari, S. & Mori, M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 11, 381–389 (2004).
Article CAS PubMed Google Scholar
Nishitoh, H. CHOP is a multifunctional transcription factor in the ER stress response. J. Biochem. 151, 217–219 (2012).
Article CAS PubMed Google Scholar
Kolb, P. S. et al. The therapeutic effects of 4-phenylbutyric acid in maintaining proteostasis. Int. J. Biochem. Cell Biol. 61, 45–52 (2015).
Article CAS PubMed Google Scholar
Muhlman, A., Lindberg, J., Classon, B., Unge, T., Hallberg, A. & Samuelsson, B. Synthesis of novel, potent, diol-based HIV-1 protease inhibitors via intermolecular pinacol homocoupling of (2S)-2-benzyloxymethyl-4-phenylbutana. J. Med. Chem. 44, 3407–3416 (2001).
Article CAS PubMed Google Scholar
Ozcan, U. et al. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 313, 1137–1140 (2006).
Article ADS PubMed PubMed Central CAS Google Scholar
Kawasaki, N., Asada, R., Saito, A., Kanemoto, S. & Imaizumi, K. Obesity-induced endoplasmic reticulum stress causes chronic inflammation in adipose tissue. Sci. Rep 2, 799 (2012).
Article ADS PubMed PubMed Central CAS Google Scholar
Ogawa, Y. et al. MHC-compatible bone marrow stromal/stem cells trigger fibrosis by activating host T cells in a scleroderma mouse model. eLife 5, e09394 (2016).
Article PubMed PubMed Central Google Scholar
Ogawa, Y. et al. Role of heat shock protein 47, a collagen-binding chaperone, in lacrimal gland pathology in patients with cGVHD. Invest. Ophthalmol. Vis. Sci. 48, 1079–1086 (2007).
Article PubMed Google Scholar
Darby, I. A. & Hewitson, T. D. Fibroblast Differentiation in Wound Healing and Fibrosis. Int. Rev. Cytol. 257, 143–179 (2007).
Article CAS PubMed Google Scholar
Kendall, R. & Feghali-Bostwick, C. A. Fibroblasts in fibrosis : novel roles and mediators. Front. Pharmacol. 5, 123 (2014).
Article PubMed PubMed Central CAS Google Scholar
Yaguchi, S. et al. Presence and physiologic function of the renin-angiotensin system in mouse lacrimal gland. Invest. Ophthalmol. Vis. Sci. 53, 5416–5425 (2012).
Article CAS PubMed Google Scholar
Alatery, A. & Basta, S. An efficient culture method for generating large quantities of mature mouse splenic macrophages. J. Immunol. Methods 338, 47–57 (2008).
Article CAS PubMed Google Scholar
Sene, A. et al. Impaired cholesterol efflux in senescent macrophages promotes age-related macular degeneration. Cell Metab. 17, 549–561 (2013).
Article CAS PubMed PubMed Central Google Scholar
Ogawa, Y. et al. Donor fibroblast chimerism in the pathogenic fibrotic lesion of human chronic graft-versus-host disease. Invest. Ophthalmol. Vis. Sci. 46, 4519–4527 (2005).
Article PubMed Google Scholar
Ogawa, Y. & Kuwana, M. Dry eye as a major complication associated with chronic graft-versus-host disease after hematopoietic stem cell transplantation. Cornea 22, 19–27 (2003).
Article Google Scholar
Kamoi, M. et al. Spontaneous lacrimal punctal occlusion associated with ocular chronic graft-versus-host disease Curr. Eye Res. 32, 837–842 (2009).
Article Google Scholar
Ogawa, Y. et al. Successful treatment of dry eye in two patients with chronic graft-versus-host disease with systemic administration of FK506 and corticosteroids. Cornea 20, 430–434 (2001).
Article CAS PubMed Google Scholar
Zhang, Y., McCormick, L. L., Desai, S. R., Wu, C. & Gilliam, A. C. Murine sclerodermatous graft-versus-host disease, a model for human scleroderma: cutaneous cytokines, chemokines, and immune cell activation. J. Immunol. 168, 3088–3098 (2002).
Article CAS PubMed Google Scholar
Nicolaou, K. C., Chakraborty, T. K., Piscopio, A. D., Minowa, N. & Bertinato, P. Total synthesis of rapamycin. J. Am. Chem. Soc. 115, 419–4420 (1993).
Google Scholar
Bachar-Wikstrom, E. et al. Stimulation of autophagy improves endoplasmic reticulum stress-induced diabetes. Diabetes 62, 1227–1237 (2013).
Article CAS PubMed PubMed Central Google Scholar
Amos, B., Smith, I. et al. A Unified Total Synthesis of the Immunomodulators (−)-Rapamycin and (−)-27-Demethoxyrapamycin: Assembly of the Common C(1–20) Perimeter and Final Elaboration. J. Am. Chem. Soc. 119, 962–973 (1997).
Article Google Scholar
Lamming, D. W., Ye, L., Sabatini, D. M. & Baur, J. A. Rapalogs and mTOR inhibitors as anti-aging therapeutics. J. Clin. Invest. 123, 980–989 (2013).
Article CAS PubMed PubMed Central Google Scholar
Salmon, A. B. About-face on the metabolic side effects of rapamycin. Oncotarget. 6, 2585–2586 (2015).
Article PubMed PubMed Central Google Scholar
Hamze, A. et al. Mono- and bis-thiazolium salts have potent antimalarial activity. J. Med. Chem. 48, 3639–3643 (2005).
Article CAS PubMed Google Scholar
Ishihara, K., Nakagawa, S. & Sakakura, A. Bulky diarylammonium arenesulfonates as selective esterification catalysts. J. Am. Chem. Soc. 127, 4168–4169 (2005).
Article CAS PubMed Google Scholar
Minamino, T., Komuro, I. & Kitakaze, M. Endoplasmic reticulum stress as a therapeutic target in cardiovascular disease. Circ. Res. 107, 1071–1082 (2010).
Article CAS PubMed Google Scholar
Yuzefovych, L. V., Musiyenko, S. I., Wilson, G. L. & Rachek, L. I. Mitochondrial DNA damage and dysfunction, and oxidative stress are associated with endoplasmic reticulum stress, protein degradation and apoptosis in high fat diet-induced insulin resistance mice. PLoS One 8, e54059 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Zeng, W. et al. 4-Phenylbutyric acid suppresses inflammation through regulation of endoplasmic reticulum stress of endothelial cells stimulated by uremic serum. Life Sci. 103, 15–24 (2014).
Article CAS Google Scholar
Galan, M., Kassan, M., Kadowitz, P. J., Trebak, M., Belmadani, S. & Matrougui, K. Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction. Biochim. Biophys. Acta. 1843, 1063–1075 (2014).
Article CAS PubMed PubMed Central Google Scholar
Ogawa, Y., Shimmura, S., Kawakita, T., Yoshida, S., Kawakami, Y. & Tsubota, K. Epithelial mesenchymal transition in human ocular chronic graft-versus-host disease. Am J Pathol 175, 2372–2381 (2009).
Article CAS PubMed PubMed Central Google Scholar
Wei, J. et al. Knockdown of thioredoxin-interacting protein ameliorates high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells. Cell. Signal 25, 2788–2796 (2013).
Article CAS PubMed Google Scholar
Dziadzio, M. et al. N-terminal connective tissue growth factor is a marker of the fibrotic phenotype in scleroderma. Q. J. M. 98, 485–492 (2005).
Article CAS Google Scholar
Oh, J. et al. Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation. J. Biol. Chem. 287, 11629–11641 (2012).
Article CAS PubMed PubMed Central Google Scholar
Xue, J. et al. Alternatively activated macrophages promote pancreatic fibrosis in chronic pancreatitis. Nat. Commun. 6, 7158 (2015).
Article ADS CAS PubMed Google Scholar
Shivshankar, P. et al. Caveolin-1 deletion exacerbates cardiac interstitial fibrosis by promoting M2 macrophage activation in mice after myocardial infarction. J. Mol. Cell. Cardiol. 76, 84–93 (2014).
Article CAS PubMed PubMed Central Google Scholar
Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. & Altman, D. G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8, e1000412 (2010).
Article PubMed PubMed Central CAS Google Scholar

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Acknowledgements

This research was subsidized by the Japanese Ministry of Education, Science, Sports and Culture, #26462668. The assistance of Dr Toshihiro Nagai, Dr Tetsuya Yano Keio University School of Medicine is also gratefully appreciated.

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Authors and Affiliations

Deaprtment of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan ,
Shin Mukai, Yoko Ogawa & Kazuo Tsubota
Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan ,
Shin Mukai, Chie Kudo-Saito & Yutaka Kawakami
Department of Medicine, Division of Endocrinology, and Department of Pathology and Immunology, Metabolism, and Lipid Research, Washington University School of Medicine, 660 S. Euclid Ave Campus Box 8127, St. Louis, MO 63110, USA.,
Fumihiko Urano

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Shin Mukai
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Yoko Ogawa
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Fumihiko Urano
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Chie Kudo-Saito
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Yutaka Kawakami
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Kazuo Tsubota
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Contributions

S.M., Y.O., K.T. conceived the study, S.M., Y.O. designed the study, S.M. performed the experiments. S.M., Y.O., C.K., F.U., Y.K., K.T. analyzed the data; S.M., Y.O., C.K., F.U., Y.K., K.T. approved the final version of this manuscript.

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Correspondence to Yoko Ogawa or Kazuo Tsubota.

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Mukai, S., Ogawa, Y., Urano, F. et al. Novel Treatment of Chronic Graft-Versus-Host Disease in Mice Using the ER Stress Reducer 4-Phenylbutyric Acid. Sci Rep 7, 41939 (2017). https://doi.org/10.1038/srep41939

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Received: 24 June 2016
Accepted: 04 January 2017
Published: 06 February 2017
DOI: https://doi.org/10.1038/srep41939

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