Accepted Manuscript PEGylated TRAIL ameliorates experimental inflammatory arthritis by regulation of Th17 cells and regulatory T cells
Jong-Sung Park, Yumin Oh, Ogyi Park, Catherine A. Foss, Sung Mook Lim, Dong-Gyu Jo, Dong Hee Na, Martin G. Pomper, Kang Choon Lee, Seulki Lee PII: DOI: Reference:
S0168-3659(17)30891-X doi:10.1016/j.jconrel.2017.10.004 COREL 8991
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
30 May 2017 2 September 2017 6 October 2017
Please cite this article as: Jong-Sung Park, Yumin Oh, Ogyi Park, Catherine A. Foss, Sung Mook Lim, Dong-Gyu Jo, Dong Hee Na, Martin G. Pomper, Kang Choon Lee, Seulki Lee , PEGylated TRAIL ameliorates experimental inflammatory arthritis by regulation of Th17 cells and regulatory T cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Corel(2017), doi:10.1016/ j.jconrel.2017.10.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Manuscript for Journal of Controlled Release
PEGylated TRAIL ameliorates experimental inflammatory arthritis by regulation of Th17 cells and regulatory T cells
IP
T
Jong-Sung Park1,2 , Yumin Oh1,2 , Ogyi Park1,2 , Catherine A. Foss1 , Sung Mook Lim3 , Dong-Gyu Jo3 ,
Rusell H, Morgan Department of Radiology and Radiological Science, 2 Center for Nanomedicine at the
US
1
CR
Dong Hee Na5 , Martin G. Pomper1,5 , Kang Choon Lee3,* and Seulki Lee1,2,5,*
Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, MD, USA School of Pharmacy, SungKyunKwan University, Suwon 16419, Korea
4
College of Pharmacy, Chung-Ang University, Seoul 06974, Korea
5
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA
ED
M
AN
3
AC
CE
PT
Correspondence to: Kang Choon Lee, [email protected]; Seulki Lee, E-mail: [email protected].
ACCEPTED MANUSCRIPT Abstract TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand that can induce apoptosis in cells expressing its cognate death receptors (DRs). Previously, we demonstrated the therapeutic potential of recombinant human TRAIL in experimental rheumatoid arthritis (RA) models. However, the
T
mechanisms of how DR-mediated apoptosis elicits these actions is not known. Here, we show that
IP
systemically administering a potent, long-acting PEGylated TRAIL (TRAILPEG) is profoundly anti-
CR
rheumatic against two complementary experimental RA mouse models, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA), via targeting IL-17 secreting Th17 cells and regulatory
US
T cells (Treg). Systemic administration of TRAILPEG after disease onset ameliorated the severity of
AN
inflammatory arthritis including arthritis indices, paw thickness, cartilage damage and neutrophil infiltration in both CIA and CAIA models. Additionally, the levels of inflammatory molecules (p-p65,
M
ICAM-1, Cox-2, MMP3, and iNOS), pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-6, IL-17)
ED
and accumulation of activated macrophages were significantly reduced after the TRAILPEG treatment. Importantly, TRAILPEG decreased the number of pro-inflammatory Th17 cells in inflamed arthritic joints
PT
through TRAIL-induced apoptosis while increasing anti-inflammatory Treg population in vivo. These
CE
results suggest that TRAILPEG ameliorates autoimmunity by targeting the Th 17 -Tregs axis, making it a
Keywords
AC
promising candidate drug for the treatment of RA.
apoptosis, autoimmune diseases, inflammation, rheumatoid arthritis, TH17-Treg, TRAIL
ACCEPTED MANUSCRIPT 1. Introduction Rheumatoid arthritis (RA) is a chronic, progressive autoimmune inflammatory disease with a 1% prevalence in the Western world [1]. Currently, there are four different categories for RA treatment: nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, non-biologic disease modifying anti-
T
rheumatic drugs (DMARDs) and biologic DMARDs. During the last twenty years, most of the RA drug
IP
development focused on DMARDs, especially biologic DMARDs. Although biological agents such as
CR
inhibitors of tumor necrosis factor (TNF) provide unseen therapeutic benefit [2], the frequency and degree of responses in patients are still restricted [3] and approximately a third of RA patients develop
US
resistance to TNF agents or experience adverse events when using them. Second -line biological drugs
AN
like rituximab can be used to treat RA that has not responded to TNF inhibitors, but these drugs are not as effective as the TNF inhibitors and may associate with serious side effects. Therefore, new disease -
M
modifying agents with improved efficacy and reduced adverse effects are necessary for better treatment
ED
of RA.
Chronic inflammation is a hallmark of RA which causes hyperplasia of synovial tissue and
PT
destruction of cartilage and bone [4, 5]. The synovial tissue from patients with RA exhibits synovial
CE
lining hyperplasia as a result of the accumulation of synovial fibroblasts, lymphocytes, and macrophage. These cells promote inflammation and joint destruction, which is mediated by the production of
AC
proinflammatory cytokines, chemokines, and proteinases [6, 7]. T cells regulate the disease process in RA on multiple levels. Regulatory T cells (Treg), which are deficient in RA, are responsible for the maintenance of peripheral immunological tolerance, whereas Th17 cells, an effector T cell, contribute to promoting inflammation by acting as Treg antagonists. In the inflamed joint, distortion of the Th17-Treg balance promotes neoangiogenesis and lymphoid organogenesis as well as stimulate synoviocyte proliferation towards autoimmune disorders [8]. Therefore, introducing a targeted agent that can
ACCEPTED MANUSCRIPT selectively maintain Th17-Treg balance without off-target toxicity is a rational and appealing approach that may reverse RA. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a death ligand that can induce apoptosis in cells expressing its cognate death receptors (DRs), DR4 and DR5 [9, 10]. Clinical
T
studies of recombinant TRAIL revealed a broad tolerability in humans but failed to demonstrate a robust
IP
therapeutic benefit in cancer patients [11], mainly due to its short half -life (less than 5 min in rodents
CR
and 30 min in humans) [12], and TRAIL-resistance in heterogeneous primary cancers [13]. To overcome the short half-life and low in vivo potency of TRAIL, we have developed a PEGylated recombinant
US
human TRAIL (TRAILPEG) [14-16]. TRAILPEG is a stabilized form of TRAIL, comprised of a zipper
AN
amino acid motif that favors trimer formation at the N -terminus with a 5 kDa poly(ethylene glycol) (PEG) molecule. TRAILPEG shows more than 16 -fold longer half-life in monkeys compared to human
M
TRAIL with similar biological activities without toxicity in primary human hepatocytes and rodents
ED
[17]. Recently, we discovered the new therapeutic potential of TRAIL in a non-cancer related disease, fibrosis. We found that systemically administered TRAILPEG ameliorates advanced liver fibrosis and
PT
cirrhosis in carbon tetra-chloride-induced liver fibrosis rat models. Based on our findings, we are
CE
interested in applying TRAILPEG in other non-cancer indications including autoimmune disease. TRAIL signaling plays an important role in maintaining B-cell and T-cell homeostasis and
AC
controlling both humoral and cellular immunity [18, 19]. In vivo, TRAIL-deficient mice are highly susceptible to RA, diabetes, experimental autoimmune encephalomyelitis (EAE), and multiple sclerosis [20-22]. TRAIL inhibits cell cycle progression and proliferation of autoreactive T cells [19] and suppresses autoimmunity by the inhibition of CD4 + cells and the promotion of Treg cells in EAE [23, 24]. These reported biological findings suggest a potential therapeutic approach to TRAIL-based therapy for autoimmune diseases. We have shown that TRAIL-based therapy can prevent progression of RA in
ACCEPTED MANUSCRIPT collagen-induced arthritis (CIA) mouse models [25, 26], however its therapeutic efficacy in RA after disease onset and the mechanisms of how TRAIL ameliorates RA have not been established. Here, we investigated the therapeutic potency of long-acting TRAIL in two complementary experimental inflammatory RA mouse models, CIA and collagen antibody-induced arthritis (CAIA), after disease
T
onset by determining clinical and histological scores and the le vels of inflammatory and pro-
IP
inflammatory markers in joint tissues after the treatment. In vivo optical imaging was applied to monitor
CR
infiltrated and accumulated macrophages in the joints of RA mice by utilizing a near -infrared imaging agent selectively targeting macrophages. Furthermore, Th17 and Treg cell populations were analyzed by
US
flow cytometry and immunohistochemistry (IHC) to explore the role of systemic TRAILPEG in RA. The
AN
results warrant further investigation into bioengineered, long-acting human TRAIL analogs as antirheumatic therapeutic strategies.
M
2. Materials and Methods
ED
2.1. Mice
DBA1/J mice (male, eight weeks; Jackson Laboratory, Bar Harbor, ME, USA) were used for all in vivo
PT
experiments and were maintained under a 12 h light/12 h dark cycle with continuous access to food and
CE
water. All mouse studies were completed under the approval and guidelines of the a nimal care and use program of Johns Hopkins University. The Johns Hopkins maintains accreditation of the program by the
International.
AC
private Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)
2.2. Preparation and characterization of TRAILPEG TRAILPEG was prepared as we previously reported [14, 15]. Briefly, human recombinant His -iLZTRAIL was reacted with 5kDa mPEG-ALD exposed in the presence of 20mM sodium cyanoborohydride (NaCNBH3 ) in 50mM sodium acetate buffer at pH 5.0. Gel-filtration chromatography
ACCEPTED MANUSCRIPT was used for recovery of TRAILPEG and concentrated by ultra-filtration and stored at -20°C. The purity of TRAILPEG is 97% as measured by FPLC (ATKA FPLC, GE Healthcare Life Science). Endotoxin levels was measured by the LAL method and confirmed
Jong-Sung Park, Yumin Oh, Ogyi Park, Catherine A. Foss, Sung Mook Lim, Dong-Gyu Jo, Dong Hee Na, Martin G. Pomper, Kang Choon Lee, Seulki Lee PII: DOI: Reference:
S0168-3659(17)30891-X doi:10.1016/j.jconrel.2017.10.004 COREL 8991
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
30 May 2017 2 September 2017 6 October 2017
Please cite this article as: Jong-Sung Park, Yumin Oh, Ogyi Park, Catherine A. Foss, Sung Mook Lim, Dong-Gyu Jo, Dong Hee Na, Martin G. Pomper, Kang Choon Lee, Seulki Lee , PEGylated TRAIL ameliorates experimental inflammatory arthritis by regulation of Th17 cells and regulatory T cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Corel(2017), doi:10.1016/ j.jconrel.2017.10.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Manuscript for Journal of Controlled Release
PEGylated TRAIL ameliorates experimental inflammatory arthritis by regulation of Th17 cells and regulatory T cells
IP
T
Jong-Sung Park1,2 , Yumin Oh1,2 , Ogyi Park1,2 , Catherine A. Foss1 , Sung Mook Lim3 , Dong-Gyu Jo3 ,
Rusell H, Morgan Department of Radiology and Radiological Science, 2 Center for Nanomedicine at the
US
1
CR
Dong Hee Na5 , Martin G. Pomper1,5 , Kang Choon Lee3,* and Seulki Lee1,2,5,*
Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, MD, USA School of Pharmacy, SungKyunKwan University, Suwon 16419, Korea
4
College of Pharmacy, Chung-Ang University, Seoul 06974, Korea
5
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA
ED
M
AN
3
AC
CE
PT
Correspondence to: Kang Choon Lee, [email protected]; Seulki Lee, E-mail: [email protected].
ACCEPTED MANUSCRIPT Abstract TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand that can induce apoptosis in cells expressing its cognate death receptors (DRs). Previously, we demonstrated the therapeutic potential of recombinant human TRAIL in experimental rheumatoid arthritis (RA) models. However, the
T
mechanisms of how DR-mediated apoptosis elicits these actions is not known. Here, we show that
IP
systemically administering a potent, long-acting PEGylated TRAIL (TRAILPEG) is profoundly anti-
CR
rheumatic against two complementary experimental RA mouse models, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA), via targeting IL-17 secreting Th17 cells and regulatory
US
T cells (Treg). Systemic administration of TRAILPEG after disease onset ameliorated the severity of
AN
inflammatory arthritis including arthritis indices, paw thickness, cartilage damage and neutrophil infiltration in both CIA and CAIA models. Additionally, the levels of inflammatory molecules (p-p65,
M
ICAM-1, Cox-2, MMP3, and iNOS), pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-6, IL-17)
ED
and accumulation of activated macrophages were significantly reduced after the TRAILPEG treatment. Importantly, TRAILPEG decreased the number of pro-inflammatory Th17 cells in inflamed arthritic joints
PT
through TRAIL-induced apoptosis while increasing anti-inflammatory Treg population in vivo. These
CE
results suggest that TRAILPEG ameliorates autoimmunity by targeting the Th 17 -Tregs axis, making it a
Keywords
AC
promising candidate drug for the treatment of RA.
apoptosis, autoimmune diseases, inflammation, rheumatoid arthritis, TH17-Treg, TRAIL
ACCEPTED MANUSCRIPT 1. Introduction Rheumatoid arthritis (RA) is a chronic, progressive autoimmune inflammatory disease with a 1% prevalence in the Western world [1]. Currently, there are four different categories for RA treatment: nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, non-biologic disease modifying anti-
T
rheumatic drugs (DMARDs) and biologic DMARDs. During the last twenty years, most of the RA drug
IP
development focused on DMARDs, especially biologic DMARDs. Although biological agents such as
CR
inhibitors of tumor necrosis factor (TNF) provide unseen therapeutic benefit [2], the frequency and degree of responses in patients are still restricted [3] and approximately a third of RA patients develop
US
resistance to TNF agents or experience adverse events when using them. Second -line biological drugs
AN
like rituximab can be used to treat RA that has not responded to TNF inhibitors, but these drugs are not as effective as the TNF inhibitors and may associate with serious side effects. Therefore, new disease -
M
modifying agents with improved efficacy and reduced adverse effects are necessary for better treatment
ED
of RA.
Chronic inflammation is a hallmark of RA which causes hyperplasia of synovial tissue and
PT
destruction of cartilage and bone [4, 5]. The synovial tissue from patients with RA exhibits synovial
CE
lining hyperplasia as a result of the accumulation of synovial fibroblasts, lymphocytes, and macrophage. These cells promote inflammation and joint destruction, which is mediated by the production of
AC
proinflammatory cytokines, chemokines, and proteinases [6, 7]. T cells regulate the disease process in RA on multiple levels. Regulatory T cells (Treg), which are deficient in RA, are responsible for the maintenance of peripheral immunological tolerance, whereas Th17 cells, an effector T cell, contribute to promoting inflammation by acting as Treg antagonists. In the inflamed joint, distortion of the Th17-Treg balance promotes neoangiogenesis and lymphoid organogenesis as well as stimulate synoviocyte proliferation towards autoimmune disorders [8]. Therefore, introducing a targeted agent that can
ACCEPTED MANUSCRIPT selectively maintain Th17-Treg balance without off-target toxicity is a rational and appealing approach that may reverse RA. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a death ligand that can induce apoptosis in cells expressing its cognate death receptors (DRs), DR4 and DR5 [9, 10]. Clinical
T
studies of recombinant TRAIL revealed a broad tolerability in humans but failed to demonstrate a robust
IP
therapeutic benefit in cancer patients [11], mainly due to its short half -life (less than 5 min in rodents
CR
and 30 min in humans) [12], and TRAIL-resistance in heterogeneous primary cancers [13]. To overcome the short half-life and low in vivo potency of TRAIL, we have developed a PEGylated recombinant
US
human TRAIL (TRAILPEG) [14-16]. TRAILPEG is a stabilized form of TRAIL, comprised of a zipper
AN
amino acid motif that favors trimer formation at the N -terminus with a 5 kDa poly(ethylene glycol) (PEG) molecule. TRAILPEG shows more than 16 -fold longer half-life in monkeys compared to human
M
TRAIL with similar biological activities without toxicity in primary human hepatocytes and rodents
ED
[17]. Recently, we discovered the new therapeutic potential of TRAIL in a non-cancer related disease, fibrosis. We found that systemically administered TRAILPEG ameliorates advanced liver fibrosis and
PT
cirrhosis in carbon tetra-chloride-induced liver fibrosis rat models. Based on our findings, we are
CE
interested in applying TRAILPEG in other non-cancer indications including autoimmune disease. TRAIL signaling plays an important role in maintaining B-cell and T-cell homeostasis and
AC
controlling both humoral and cellular immunity [18, 19]. In vivo, TRAIL-deficient mice are highly susceptible to RA, diabetes, experimental autoimmune encephalomyelitis (EAE), and multiple sclerosis [20-22]. TRAIL inhibits cell cycle progression and proliferation of autoreactive T cells [19] and suppresses autoimmunity by the inhibition of CD4 + cells and the promotion of Treg cells in EAE [23, 24]. These reported biological findings suggest a potential therapeutic approach to TRAIL-based therapy for autoimmune diseases. We have shown that TRAIL-based therapy can prevent progression of RA in
ACCEPTED MANUSCRIPT collagen-induced arthritis (CIA) mouse models [25, 26], however its therapeutic efficacy in RA after disease onset and the mechanisms of how TRAIL ameliorates RA have not been established. Here, we investigated the therapeutic potency of long-acting TRAIL in two complementary experimental inflammatory RA mouse models, CIA and collagen antibody-induced arthritis (CAIA), after disease
T
onset by determining clinical and histological scores and the le vels of inflammatory and pro-
IP
inflammatory markers in joint tissues after the treatment. In vivo optical imaging was applied to monitor
CR
infiltrated and accumulated macrophages in the joints of RA mice by utilizing a near -infrared imaging agent selectively targeting macrophages. Furthermore, Th17 and Treg cell populations were analyzed by
US
flow cytometry and immunohistochemistry (IHC) to explore the role of systemic TRAILPEG in RA. The
AN
results warrant further investigation into bioengineered, long-acting human TRAIL analogs as antirheumatic therapeutic strategies.
M
2. Materials and Methods
ED
2.1. Mice
DBA1/J mice (male, eight weeks; Jackson Laboratory, Bar Harbor, ME, USA) were used for all in vivo
PT
experiments and were maintained under a 12 h light/12 h dark cycle with continuous access to food and
CE
water. All mouse studies were completed under the approval and guidelines of the a nimal care and use program of Johns Hopkins University. The Johns Hopkins maintains accreditation of the program by the
International.
AC
private Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)
2.2. Preparation and characterization of TRAILPEG TRAILPEG was prepared as we previously reported [14, 15]. Briefly, human recombinant His -iLZTRAIL was reacted with 5kDa mPEG-ALD exposed in the presence of 20mM sodium cyanoborohydride (NaCNBH3 ) in 50mM sodium acetate buffer at pH 5.0. Gel-filtration chromatography
ACCEPTED MANUSCRIPT was used for recovery of TRAILPEG and concentrated by ultra-filtration and stored at -20°C. The purity of TRAILPEG is 97% as measured by FPLC (ATKA FPLC, GE Healthcare Life Science). Endotoxin levels was measured by the LAL method and confirmed
