RIPK1 inhibitor ameliorates colitis by directly maintaining intestinal barrier homeostasis and regulating following IECs-immuno crosstalk

Journal Pre-proofs RIPK1 inhibitor ameliorates colitis by directly maintaining intestinal barrier homeostasis and regulating following IECs-Immuno crosstalk Huimin Lu, Heng Li, Chen Fan, Qing Qi, Yuxi Yan, Yanwei Wu, Chunlan Feng, Bing Wu, Yuanzhuo Gao, Jianping Zuo, Wei Tang PII: DOI: Reference:

S0006-2952(19)30450-2 https://doi.org/10.1016/j.bcp.2019.113751 BCP 113751

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Biochemical Pharmacology

Received Date: Accepted Date:

11 October 2019 9 December 2019

Please cite this article as: H. Lu, H. Li, C. Fan, Q. Qi, Y. Yan, Y. Wu, C. Feng, B. Wu, Y. Gao, J. Zuo, W. Tang, RIPK1 inhibitor ameliorates colitis by directly maintaining intestinal barrier homeostasis and regulating following IECs-Immuno crosstalk, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.113751

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Title page: RIPK1 inhibitor ameliorates colitis by directly maintaining intestinal barrier homeostasis and regulating following IECs-IMMUNO crosstalk Huimin Lu1,2, Heng Li1,2, Chen Fan1, Qing Qi1, Yuxi Yan1,2, Yanwei Wu1, Chunlan Feng1, Bing Wu1,2, Yuanzhuo Gao1,2, Jianping Zuo1,2*, Wei Tang1,2*

Affiliations:1 Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China 2 University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China

* Corresponding author Jianping Zuo Laboratory of Immunopharmacology State Key Laboratory of Drug Research Shanghai Institute of Materia Medica, Chinese Academy of Sciences 555 Zuchongzhi Road, Zhangjiang, Shanghai 201203, China Tel.: 86-21-50806701 E-mail: [email protected] Wei Tang Laboratory of Immunopharmacology State Key Laboratory of Drug Research Shanghai Institute of Materia Medica, Chinese Academy of Sciences 555 Zuchongzhi Road, Zhangjiang, Shanghai 201203, China Tel.: 86-21-50806820 E-mail: [email protected]

Acknowledgements This work was funded by the "Personalized Medicines—Molecular Signature-based Drug Discovery and Development", Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA12020370) and the Science & Technology Commission of Shanghai Municipality, China (Grant No.18431907100).

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1

RIPK1 inhibitor ameliorates colitis by directly maintaining intestinal barrier

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homeostasis and regulating following IECs-IMMUNO crosstalk

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Abstract

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Background: The receptor-interacting protein kinase 1 (RIPK1) has emerged as a key upstream

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regulator that controls the inflammatory response via its kinase-dependent and independent

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functions, which makes it an attractive target for developing new drugs against

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inflammation-related diseases. Growing evidences illustrate that RIPK1 is certainly associated

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with pathogenesis of multiple tissue-damage diseases. However, what are intricate regulatory

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codes of RIPK1 inhibitor in diseases is still obscure.

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Methods: We used DSS-induced colitis model in vivo to study the therapeutic effects and the

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mechanisms of RIPK1 inhibitor. We next characterized the barrier function and the interaction

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between intestinal epithelial cells (IECs) and immunocytes both in vivo and in vitro. As a

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candidate in clinical study, GSK2982772 is the most well-developed drug of RIPK1 inhibitors,

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and we chose it as our study object.

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Results: We demonstrated that RIPK1 inhibitor could ameliorate the intestinal barrier injury by

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reducing tight junctions’ disruption and accompanying oxidative stress. Moreover, the release of

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chemokines and adhesion molecules from damaged IECs was suppressed by RIPK1 inhibitor

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treatment. And these protective effects were not only dependent on the suppression of necroptosis

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but also on the compromised activity of NF-κB. Taken together, RIPK1 inhibitor exerts

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suppressive function in intestinal inflammatory response possibly via protecting the intestinal

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epithelial barrier and maintaining the homeostasis of immune microenvironments. Eventually, the

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positive feedback immune response which triggered progressive epithelial cells injury could be

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restrained.

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Keywords: RIPK1 inhibitor; Colitis; Barrier homeostasis; IECs-immunocytes crosstalk;

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necroptosis; NF-κB;

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1.

Introduction

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The receptor-interacting protein kinase 1 (RIPK1), has emerged as an important upstream

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kinase which could affect multiple cellular pathways associated with regulating inflammation.

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While, there is controversy about the feature of RIPK1 in the pathogenesis of colitis. For one side,

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current studies deem that RIPK1 has a pathogenic mechanism in colitis due to epithelial

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necroptosis[1, 2], which process is dependent on the its kinase activity. The activated RIPK1 could

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recruit RIPK3 and MLKL to form necrosome to contribute proinflammatory response[3]. For

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another, in RIPK1 deficient mice, the essential protective effects of RIPK1 in intestinal

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homeostasis reside in its kinase-independent scaffold function, which can inhibit RIPK3-mediated

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necroptosis by RIP homotypic interaction motif (RHIM)[4-6]. However, without genetic

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intervention, evidence about the role of RIPK1 in human inflammatory bowel disease (IBD)

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remains limited[3]. The exact contribution of RIPK1 in colitis is an indispensable question for

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drug development. It is worth mentioning that the inhibitors targeting RIPK1 are implementing in

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pharmacodynamics studies of inflammatory diseases. Currently, GSK2982772 is in multiple Phase

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2 clinical trials to treat inflammatory diseases [7]. However, the pharmacological mechanism of it

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has not been reported and the effects of RIPK1 inhibitor in colitis have abundant room for further

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progress in determining.

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Ulcerative colitis (UC) is an inflammatory bowel disease characterized by mucosal barrier

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damage and immune cells infiltration [8]. The intact barrier of intestinal could defense the

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invasion of pathogens and antigens[9]. In pathologic states, a leaky epithelial barrier results in

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excessive exposure to microbial antigens, recruitment of immune cells, release of inflammatory

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mediators, and eventually leading to progressive intestinal mucosa injury [10]. The integrity of the

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epithelial barrier largely depends on intestinal epithelial cells (IECs) and intercellular junctions

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that include tight junctions (TJ) [9].

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As an indispensable component of the mucosal barrier, IECs are of great importance on

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maintaining barrier integrity and homeostasis. Some reports about the relationship between RIPK1

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function and IECs homeostasis were limited to necroptosis, which lead to loss of integrity of

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intestinal barrier[11]. Meanwhile, dying cells indirectly trigger inflammation by releasing

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damage-associated molecular patterns (DAMPs)[3]. Besides necroptosis, there may be some

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interesting mechanisms involve in the effects of RIPK1 in mediating barrier structural and 3

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functional disorders. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction

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and a potential therapeutic target. Intraperitoneal TNF administration can induce barrier loss and

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enhance intestinal epithelial MLC phosphorylation[12]. Further work is required to establish

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whether there is a pertinence between RIPK1 and MLC phosphorylation. Moreover, Reactive

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oxygen species (ROS) is generally labeled as a proinflammatory factor, and its overproduction has

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been strongly related to Crohn’s disease and pancolitis[13].The oxidative stress of IECs and the

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action of RIPK1 in this process are deserved to study.

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Finally, in consideration of the limited reports about the immunosuppressive function of

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RIPK1 inhibitor on immunocytes, it is a novel point to focus on the pharmacological mechanisms

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of RIPK1 inhibitor in IECs and following IECs-immunocytes crosstalk. Delicate and precise

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interactions between epithelial cells and immune cells determine mucosal homeostasis. Even a

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slight deviation might lead to epithelial barrier injury, translocation of luminal antigens and

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immune responses[9]. Intercellular adhesion molecules (ICAMs) and chemokines generated by

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epithelial cells could recruit immune cells and have become integral in matters of crosstalk

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between epithelial cells and immunocytes. Elevated ICAMs are identified as trigger factors for

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immunocytes adhesion, migration and local lymphocytes activation, and are responsible for

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immunocytes trafficking in the focal inflammation. Epithelial ICAM-1 is related to inflammation

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response by contributing to immune cells migration to peri-epithelial sites, and its blockade has

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become a therapeutic target for colitis[14, 15]. Chemokines and their receptors as well as perform

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indispensable functions in orchestrating tissue-specific leukocytes trafficking[16]. There is a

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growing body of clinical data that the elevated levels of chemokines and their receptors were

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observed in patients with IBD, and decreasing their production may help to ameliorate

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diseases[17-19]. There have arisen several novel drugs target chemokines to treat colitis in clinical

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trials and showed promising therapeutic results[20]. It is meaningful to explore the RIPK1-related

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adhesion and chemotaxis behavior of leukocytes trafficking to epithelial cells, due to a noticeable

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role of the crosstalk between IECs and immunocytes in colitis etiology.

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In this study, we developed the murine colitis model to explore the possible mechanisms how

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RIPK1 inhibitor protects barrier injury and interferes with the interaction between IECs and

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immunocytes. 4

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2.

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2.1. Reagents and antibodies

Materials and methods

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GSK2982772, a RIPK1 inhibitor, was purchased from Med Chem Express (NJ, USA) and

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dissolved in dimethyl sulfoxide (DMSO) as a stock solution. Dextran Sulfate Sodium (DSS,

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molecular weight 36-50 kDa) was purchased from MP Biomedicals (Irvine. CA, USA).

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(Hydroxypropyl)methyl cellulose (HPMC) was purchased from Sigma-Aldrich (St Louis, MO,

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USA). Recombinant human TNF-α was obtained from Peprotech (London, UK). Fecal occult

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blood test kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

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RIPK3i (GSK872), MLKLi (NSA), NF-κBi (TPCA-1), SM164 and ZVAD were purchased from

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Med Chem Express (NJ, USA). N-acetyl-cysteine was purchased from Sigma-Aldrich (St Louis,

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MO, USA).

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Thermo Scientific (Pittsburgh, PA, USA). FITC-dextran was purchased from Sigma (St Louis,

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MO, USA). Cell Titer-Glo Luminescent Cell Viability Assay was purchased from Promega

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(Madison, USA). Reactive Oxygen Species Assay Kit was purchased from Beyotime (Haimen,

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China). Calcein AM was purchased from Abcam (Cambridge, UK). In Situ Cell Death Detection

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Kit (TUNEL) was purchased from Roche Diagnostics GmbH (Mannheim, Germany).

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Proteinase and phosphatase inhibitor and BCA assay kit were purchased from

Anti-mCD16/CD32,

PE-anti-F4/80,

PE-anti-Ly6G

PE-anti-CD25,

PE-anti-CD278,

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PE-anti-IL-17,

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APC-anti-Ly6c, APC-anti-IFNγ were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

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ELISA kits for mouse IL-17, IL-1β, IL-6, TNF-α were purchased from eBioscience (San Diego,

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CA, USA). Human IL-8, CXCL10 ELISA kits were purchased from Biolegend (San Diego, CA,

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USA). Anti-p-RIPK1, Anti-p-RIPK3, Anti-ICAM1, Anti-IκBα, Anti-p-P65, Anti-p-MLCK and

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anti-Ecadherin were obtained from Cell Signaling (Danvers, MA, USA). Anti-mICAM1,

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Anti-CD3, Anti-HSP90 and FITC-anti-CD11b were purchased from Abcam (Cambridge, UK).

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Anti-Zo-1 and Anti-p65 were purchased from Proteintech (Chicago, IL, USA). Anti-Occludin was

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purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-HMGB1 was purchased

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from Serotec (Oxford, UK).

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2.2. Cell culture

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PercP-Cy5.5-anti-CD11b,

PercP-Cy5.5-anti-CD69,

APC-anti-γδTCR,

The human colonic adenocarcinoma cell line HT-29, Caco-2 cells, the human acute 6

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monocytic leukemia cell line THP-1 cells and T lymphocyte cell line Jurkat cells were purchased

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from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were culture din

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McCoy's 5a Medium, DMEM and RPMI 1640 medium (Gibco, Grand Island, NY, USA),

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respectively, containing 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/ml penicillin

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and 100 µg/ml streptomycin. Cells were cultured in a humidified incubator with 5% CO2 at 37°C.

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2.3. Mice

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Female C57BL/6J mice (6-8 weeks, 18-20 g) were obtained from Shanghai Lingchang

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Biotechnology Co.Ltd. (certificate no. 2013-0018, shanghai, China), and were maintained at the

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specific pathogen-free (SPF) animal facilities of Shanghai Institute of Materia Medica. All

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experiments were performed on the basis of the guidelines of the Association Assessment and

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Accreditation of Laboratory Animals Care International. And all of the procedures were carried

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out strictly in accordance with the animal care and use protocol (2018-03-TW-06) approved by the

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Institutional Animal Care and Use Committee (IACUC) at Shanghai Institute of Materia Medica.

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2.4. Models of colitis

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Acute colitis was achieved by feeding of 3% (w/v) DSS (MP Biomedicals, molecular mass

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36,000-50,000 Da) in drinking water. Mice were received either regular drinking water (normal

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control and normal+RIPK1i) or DSS drinking water (vehicle and RIPK1i) for 6 days followed by

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3 days of regular drinking water. Mice were randomly divided into 4 groups with 10 mice per

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group: normal control (HPMC), Normal+RIPK1i (GSK2982772, 20 mg/kg), vehicle control

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(HPMC) and RIPK1i (GSK2982772, 20 mg/kg). Mice were orally administered once daily for 9

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days and sacrificed at day 10. (Fig 1A).

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Additionally, we performed an independent experiment and sacrificed mice at disease active

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stage (Day6) for observing the treatment effect of RIPK1i in acute phase (figure 8A).

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2.5. Clinical assessment of colitis

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For monitoring the severity of colitis, body weight, stool consistency and rectal bleeding

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were assessed daily. Values assessed prior to DSS exposure served as baseline. Weight changes

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were calculated in relation to the weight at baseline (100%). And the disease activity index (DAI)

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was calculated based on the scoring system[21].

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2.6. Histology 7

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Intestinal tissues were fixed in 4% paraformaldehyde, embedded in paraffin. For

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histopathological analysis, hematoxylin and eosin staining was performed according to standard

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protocol. Histological evaluation of H&E-stained colonic sections was achieved by two

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independent observers blinded to the experimental conditions and graded as previously

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described[22]. For immunofluorescence colonic tissues were embedded in OCT compound, and

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sectioned on a cryostat (6 µm thick). After fixed in paraformaldehyde, sections were blocked with

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5% BSA for 60 min and then incubated with FITC-anti-CD11b (Abcam) at 4°C overnight. The

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sections were counterstained with 4', 6-diamidino-2-phenylindole (DAPI) (Abcam). Fluorescent

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sections were visualized and images were captured using a Leica TCS SP8 STED confocal

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microscope. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded

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tissues with rabbit anti-CD3(Abcam), rat anti-HMGB1 (Serotec) antibodies. These sections

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microscopy was performed using a Leica DM 6B microscope.

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2.7. Immunofluorescence cytochemistry

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HT-29 cells on coverslips were fixed in fixing solution (Beyotime, China) for 15 min. After

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treated blocking buffer (Beyotime) for 60 min, cells were incubated with rabbit anti-Ecadherin

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(Cell signaling), Zo-1(Proteintech), Occludin (Thermo) respectively overnight at 4°C. The FITC

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or PE-conjugated anti-rabbit secondary antibodies (Proteintech, Rosemont, USA) was added, after

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washing with 1% PBS-Tween. Negative control reactions were included in each experiment and

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carried out by replacing primary antibodies with PBS. The cells were counterstained with DAPI.

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All images were captured using a Leica TCS SPS CFSMP microscope.

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2.8. TdT-mediated dUTP Nick-End Labeling (TUNEL)

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Cell death was analyzed with an in-situ cell death detection kit (TMR-red, Roche).

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Fluorescence microscopy was performed using a Leica TCS SPS CFSMP microscope.

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2.9. Assessment of myeloperoxidase (MPO) activity

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For measured the neutrophil infiltration into inflamed colonic mucosa, MPO activity was

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detected by O-dianisidine method as previously described[23]. The results were showed as activity

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units per gram tissue.

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2.10.

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Single cell preparation and Flow cytometry analysis For single cell suspension preparation, spleens and mesenteric lymph nodes (MLNs) from 8

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mice were grinded and filtered through a 40 µm nylon mesh strainer. Colons were cut into small

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pieces after removing intestinal contents and residual fat. And turned the tissue inside out by

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cannulating the intestinal segments with curved forceps. Cleaned colon fragments were incubated

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in extraction media (30 ml RPMI + 93 µl 5% (w/v) dithiothreitol (DTT) (Meilun Biotechnology,

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Dalian, China)+ 60 µl 0.5 M EDTA (Sigma, USA))+ 500 µl fetal bovine serum (FBS)) for 15min

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on constant temperature shaker (600 rpm) to dissociate epithelial cells, and then added the minced

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small intestine to a centrifuge tube containing 25 ml of digestion media (25 ml RPMI + 12.5 mg

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dispase + 37.5 mg collagenase II + 300 µl FBS) for incubating at 500 rpm for 30 min at 37 °C.

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Filter digested tissue through a 100 µm cell strainer into a 50 ml tube. After centrifugation, filter

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resuspended cells through a 40 µm cell strainer into a 50 ml tube. Finally, resuspend pellet in 1 ml

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of RPMI containing 2% FBS.

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The single-cells unpensions were blocked with anti-mCD16/CD32 (2.4G2) and then stained

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with antibodies in dark. Flow cytometric analysis was performed on BD LSR Fortessa, and data

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were analyzed using FlowJo 10 software (Treestar, Ashland, OR).

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2.11.

FITC–dextran intestinal permeability assay

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Intestinal permeability was assessed by oral gavage of FITC–dextran 4KD (Sigma), a

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macromolecule that cannot be metabolized and is used as a permeability probe. Mice were

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administered 200 μl FITC–dextran (600mg/kg bodyweight) by oral gavage 4h before killing.

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Whole blood was obtained by removing eyeball, and FITC–dextran levels of serum were

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measured by fluorometry (Ex:488 nm, Em:525 nm). For observing permeable fluorescence signal

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directly, mice were imaged in vivo by PE IVIS spectrum (Perkin Elemer).

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In vitro, FITC-dextran permeability assays were performed as previously described[24]. In

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brief, 5x104 Caco-2 cells were seeded onto 0.4μm pore size trans-well inserts (Corning, NY, USA)

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and grown to confluence. After hTNFα (100ng/ml) treatment for 24 hours, 10 μg/mL

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FITC-dextran 4KD solution was added to the inserts. After allowing 2h for diffusion, the medium

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from the lower chamber was collected and analyzed on a fluorescence plate reader.

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2.12.

Transepithelial electrical resistance (TEER)

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To measure the intestinal barrier function, 5x104 Caco-2 cells were seeded on the apical side

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of 0.4μm pore size transwell polyester membrane filters,(Corning, NY, USA), and integrity of cell 9

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monolayers was determined with trans epithelial electrical resistance (TEER) using an epithelial

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Volt-Ohm Meter (Millicell ERS2)[25].

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2.13.

In vitro leukocytes adhesion

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HT-29 cells plated on 24-well plates were treated with RIPK1i (50nM) for 30min and then

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stimulated with TNFα (100ng/ml) for 24 h. THP-1 monocytes (2x105 cells/mL) were fluorescently

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labeled with 2.5 μg/mL Calcein AM (Abcam) in RPMI-1640 medium for 30min. After washing

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twice with PBS, Calcein AM-labeled THP-1 cells were resuspended in fresh RPMI-1640 medium

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and then added at 105 cells/well onto a HT-29 monolayer and incubated at 37℃ for 30 min.

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Non-adherent THP-1 cells were washed away by PBS. The representative image of adherent

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THP-1 cells were captured under a fluorescence microscope (Olympus IX73)[26].

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2.14.

Chemotaxis assay

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HT-29 cells were treated with RIPK1i (50nM) for 30min and then stimulated with TNFα

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(100ng/ml) for 24 h. Subsequently, conditional medium (600μl) were collected and added into the

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lower chamber of 24-well trans-well chambers with 8 µm pores (Corning, NY, USA). THP-1 and

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Jurkat cells (1 × 106 cells/mL) were labeled with 2.5 μg/mL Calcein AM in RPMI-1640 medium

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for 30min. After washing twice with PBS, THP-1 and Jurkat cells were resuspended in fresh

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RPMI-1640 medium and then added at100μl /well onto upper chamber of trans-well for 2 or 4 h

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incubated at 37℃ respectively. The number of cells in lower chamber was detected by cytometer

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and fluorescence microscope (Olympus IX73)[27].

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2.15.

Quantitative real-time PCR

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Total RNA was isolated from HT-29 cells and colonic tissues by using RNA simple total

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RNA kit (Tiangen, Beijing, China). Then total RNA was reverse transcribed by an All-in-One

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cDNA Synthesis SuperMix (Biotool, Houston, TX, USA). Quantitative real-time PCR was

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performed with SYBR® Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan) on a 7500

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Fast Real-Time PCR System (Applied Biosystems, Foster city, CA, USA).

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2.16.

Cytokine analysis by ELISA

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Colons from mice were homogenized with lysis buffer to extract total protein as described by

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Janice J. Kim et al [23]. The concentration of total protein was determined by BCA protein assay

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kit. Cytokines level of TNF-α, IL-1β, IL-6, IL-17 in colon homogenate and chemokines level of 10

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CXCL10, IL-8 in HT-29 cell culture supernatant were quantified by ELISA kit (Biolegend).

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2.17.

Western blot analysis

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The colon tissues were homogenized and HT-29 cells were lysed in sodium dodecyl sulfate

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lysis buffer (Beyotime) containing proteinase and phosphatase inhibitor. The protein

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quantification was determined by BCA Protein Assay Kit (Thermo Scientific) The equal amounts

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of proteins were separated on SDS-polyacrylamide gel electrophoresis and transferred to a

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nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). After blocking,

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the membranes were incubated with primary antibodies overnight at 4 ℃. After washing with TBS

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with Tween-20, the secondary antibodies (1:20000, Bio-Rad, Richmond, CA, USA) were added,

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and HRP-conjugated monoclonal mouse anti-GAPDH (1:10000, Kangcheng, Shanghai, China) as

242

control for normalization. Signals were detected with ECL system (Amersham Bioscience,

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Buckinghamshire, UK) and exposed to classic autoradiography film or Amersham Imager 600

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(GE).

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2.18.

Statistical analysis

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Data were presented as mean ± SEM and all group data subjected to statistical analysis in the

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present research have a minimum of n=3 individuals per group (the value of n is indicated in the

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specific legend). The results of western blot, flow cytometry and morphology analysis were

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presented as representative images. Statistical analyses were conducted using GraphPad Prism 5.0

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software. All experiments were repeated at least three times, with similar results. Significant

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differences between groups were determined using a one-way ANOVA with Dunnet’s multiple

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comparisons test with no significant variance inhomogeneity (F achieved p<0.05) and p<0.05 was

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considered to represent a significant difference.

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3.

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3.1. RIPK1 inhibitor ameliorated DSS-induced colitis and suppressed the proinflammatory cytokines and DAMPs production

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In order to confirm the pharmacological action of RIPK1 inhibitor in colitis, Dextran Sulfate

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Sodium (DSS) induced acute colitis model was performed (Figure 1A). To get more information

259

about the possible toxicity, the naïve mice were treated with the RIPK1 inhibitor. The evaluated

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indicators, including body weight, DAI, colon length, serum ALT and ALP and pathological

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sections, suggested that RIPK1 inhibitor treatment has no adverse events in indicated dose. (Figure

Results

11

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1B-E). Sustained body weight loss and disease activity index (DAI) rise were relieved to a certain

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extent during RIPK1 inhibitor (RIPK1i, GSK2982772 20mg/kg) administration (Figure 1B).

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Consistently, the colon shortening was restored in RIPK1i-treatment group (Figure 1C). The

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serum ALT and ALP, which indicate the liver function, showed improvement under RIPK1i

266

treatment (Figure 1D). The severity of histology disruption was analyzed by H&E staining.

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Representative microscopic images and histopathological scores of colonic sections reflected the

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improvement effect of RIPK1 inhibitor (Figure 1E). As expected, the phosphorylation level of

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RIPK1 did increase in the vehicle group, which indicated the pathogenetic role of RIPK1

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activation in colitis (Figure 1F). Increased release of proinflammatory cytokines and DAMPs

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contributed to the development of colitis. RIPK1 inhibition could suppress these cytokines levels

272

both in serum and colonic tissue (Figure 1G). The production of high mobility group box1

273

(HMGB1) and heat shock protein (HSP90) also was suppressed by RIPK1 inhibitor treatment

274

(Figure 1H).

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3.2. RIPK1 inhibitor suppressed the immune response in colitis but had few direct effects on

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immunocytes in vitro

277

The secondary immune organs, such as spleen and mesenteric lymph nodes, can be driven to

278

exert immune defense functions with the development of colitis. However, aberrant immunocytes

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activation can deteriorate the progression of disease. We prepared a single cell suspension isolated

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from spleen and mesenteric lymph nodes to test the marker of T cell activation, including CD25,

281

CD69 and CD278 gated from CD3 and CD4 positive cellular population. Compared to the vehicle

282

controls, RIPK1 inhibitor-treated mice showed an obvious reduction in the percentage of activated

283

T lymphocytes. (Figure 2A). It has been reported that γδT cells are involved in the exacerbation of

284

colitis[28]. In colitis model, we observed the increase of γδT cells in spleen can be controlled by

285

RIPK1 inhibitor administration. (Figure 2B). Blockage the differentiation of Th17 cells could

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ameliorate colitis[29]. In our study, RIPK1 inhibition intervened the differentiation of Th17 cells

287

in spleen (Figure 2C). In conclusion, inhibition of RIPK1 delayed the immune response by

288

modulating the activation and differentiation of T cells in immune organs. Furthermore, we found

289

that RIPK1 inhibitor could reduce a high level of innate immunocytes both in mesenteric lymph

290

nodes and spleen, such as neutrophil (CD11b+ly6c+/ly6G+) and macrophage (CD11b+F4/80+) 12

291

(Figure 2D). To determine whether RIPK1 inhibitor has direct immunosuppressive effects on

292

immunocytes, we prepared spleen lymphocytes to investigate the suppressive function of RIPK1

293

inhibitor on the proliferation status, which stimulated by Concanavalin (ConA) and LPS. The

294

results suggested that RIPK1 inhibitor had the comparable value of CC50 and IC50. RIPK1 kinase

295

inhibition may have negligible effects on lymphocytes activation (figure 3A and B). Furthermore,

296

we performed relative experiments on bone marrow-derived macrophage (BMDM) and bone

297

marrow-derived dendritic cells (BMDC). The results revealed that RIPK1 and necroptosis

298

pathway inhibition had no effects on LPS-induced cytokines elevation both in BMDM and BMDC

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(figure 3C and D). Taken together, RIPK1 inhibitor may have inapparent effects on immunocytes

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function.

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3.3. RIPK1 inhibitor restrained the immunocytes infiltration by maintaining intestinal barrier

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homeostasis and chemotaxis process in colitis

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According to the above data, we can infer that RIPK1 inhibitor may play a protective role in

304

the early stages of disease progression. Before the systemic immune response, there will be many

305

immunocytes infiltration in the lamina propria of the colon. In immunohistochemical results, the

306

infiltration of CD3 positive cells increased significantly in the vehicle group, and RIPK1 inhibitor

307

could relieve this symptom (Figure 4A). As shown in Figure 4B, the decreased Treg

308

(CD25+Foxp3+) and increased Th17 (IL-17+) in the lamina propria could be restored by this

309

inhibitor. Myeloperoxidase (MPO), which is most abundantly expressed in neutrophil

310

granulocytes, showed an enhanced activity in colon tissue of colitis mice. This indicated that

311

RIPK1i treatment may reduce the massive neutrophil infiltration in colon. (Figure 4C).

312

Correspondingly, CD11b expression was detected in situ, which can reflect the infiltration of

313

myeloid immune cells. The result of myeloid immune cells infiltration was consistent with the

314

above (Figure 4D). To confirm this event, we prepared a single cell suspension isolated from

315

colon lamina propria to determinate the percentage of infiltrated cells by flow cytometry. A large

316

number of accumulating neutrophils (CD11b+Ly6G+) and macrophages (CD11b+F4/80+) was

317

lowered in lamina propria after RIPK1 inhibitor treatment compared with the vehicle (Figure 4E).

318

In view of the epithelium barrier as a defense system against pathogens, RIPK1 inhibitor may

319

have a positive role in barrier function and then reduce the infiltration of immunocytes in colon. 13

320

We next used the TUNEL assay to visually detect intestinal epithelium injury in situ. Inhibition of

321

RIPK1 obviously lessened epithelium injury in colitis (Figure 4F). The expression of tight

322

junction proteins, including E-cadherin, Zo-1 and Occludin was restored in RIPK1i-treatment

323

group (Figure 4G), indicating the protective effects of RIPK1 inhibition in maintaining barrier

324

integrity. Intestinal permeability was assessed by oral gavage of FITC–dextran. Both in serum

325

detection and in vivo imaging, we all found that the absorption of fluorescence was lower in

326

RIPK1i-treatment group compared with the vehicle (Figure 4H), which demonstrated that RIPK1

327

inhibitor preserved the intact intestinal barrier.

328

IECs-immunocytes interactions, as well as inflammatory mediators release during this

329

process, are critical pathogenic factors in colitis. Therefore, we paid attention to the role of RIPK1

330

inhibitor in this pathological process. In colitis model, results suggested that the expression of

331

chemokines and its receptors in colon was RIPK1 dependent. This result has also been observed in

332

the level of ICAM1 (Figure 4I and J). This finding, while preliminary, suggests that RIPK1

333

inhibitor protects the structure and function of the intestinal epithelial barrier and reduces the

334

secretion of chemokines and adhesion molecules from IECs, then lowering the infiltration of

335

immunocytes.

336

3.4. RIPK1 inhibitor maintained IECs homeostasis by alleviating cell death, MLCK-related

337

tight junctions’ injury and accompanying oxidative stress

338

Firstly, epithelial cells viability is essential for the integrity of intestinal barrier. The results

339

suggested that TSZ (TNF+SM164+ZVAD) treatment did induce evident HT-29 cell death, and

340

RIPK1 inhibitor restored this injury (Figure 5A). As shown in Figure 5B, cell death did not occur

341

obviously under TNF stimulation alone. If RIPK1 inhibitor has a protective mechanism in IECs

342

besides of cell death? To answer this question, we explored whether RIPK1 inhibitor can protect

343

the injury of tight junctions in IECs, which as an indispensable factor in maintaining epithelium

344

integrity. The results suggested that RIPK1 inhibition ameliorated TNFα-triggered destruction of

345

E-cadherin, Zo-1 and Occludin (Figure 5C). To further verify the effect of RIPK1 inhibitor on

346

functional profile of IECs, we detected the permeability of colonic epithelial cell, Caco-2. The

347

FITC-Dextran flux through monolayers was elevated with TNFα incubation and RIPK1 inhibition

348

alleviated this high permeability (Figure 5D). The epithelial monolayers have a certain value of 14

349

trans epithelial electrical resistance (TEER). When barrier structural disturbances, the drop of

350

TEER value could be tested. RIPK1 inhibitor treatment rescued this compromise of TEER

351

triggered by TNFα (Figure 5E). Some researches showed that MLCK activation resulted in

352

dysregulation of tight junctions, barrier loss and induction of colitis[12, 30]. We found that RIPK1

353

was related to MLC phosphorylation in HT-29 cells (Figure 5F). RIPK1 inhibitor may play a

354

protective effect in IECs by suppressing the activation of MLCK. Moreover, RIPK1 inhibition

355

could reduce the ROS release of IECs, and it is RIPK3 dependent (Figure 5G).

356

3.5. RIPK1 inhibitor could influence the interaction between IECs and immunocytes by

357

suppressing the production of chemokines and adhesion molecules of epithelium

358

Based on the RIPK1 inhibition could reduce the immunocytes infiltration in intestinal lamina

359

propria, we would like to explore IECs-immunocytes crosstalk in vitro. The results showed that

360

RIPK1 inhibitor had evident effects on down-regulation of chemokines and ICAM1 genes

361

expression of HT-29 cells (Figure 6A). To test the protein expression level, we detected the IL-8

362

and CXCL10 secretion by ELISA. As shown in (Figure 6B), RIPK1 inhibition suppressed the high

363

level of chemokines in response to TNFα. The expression of ICAM-1 also was inhibited

364

significantly at different time points during this process (Figure 6C). Functionally, enhanced

365

adhesive ability of HT-29 cells was observed under the stimulation of TNFα and RIPK1 inhibition

366

intervened this event (Figure 6D). In accordance with the decreased chemokines level after RIPK1

367

inhibition, the chemotaxis behavior of THP-1 and Jurkat cells to conditional media from HT-29

368

cells was restrained by RIPK1 inhibitor treatment (Figure 6E and F). We thought that RIPK1

369

inhibition suppressed the chemotaxis and adhesion processes of immunocytes towards IECs.

370

RIPK1 may participate in the interaction between IECs and immunocytes, which possibly

371

becomes one of the underlying mechanisms of RIPK1 mediating barrier injury-related

372

inflammation.

373

3.6. The suppressive effect of RIPK1 inhibitor on cytokines of IECs was related to necroptosis

374

and NF-κB pathway

375

We would be interested to know what mechanisms involved in the suppressive function of

376

RIPK1 inhibitor on the production of chemokines and ICAM1 from IECs. Firstly, we found that

377

RIPK1 inhibitor could reduce the release of cytokines both under TNF and TSZ stimulation, 15

378

which represented necroptosis and non-cell death conditions, respectively (Figure 7A). For

379

necroptosis process, RIPK1 inhibitor prevented the activation of necrosome (p-RIPK3 and

380

p-MLKL). And then the production of pro-inflammatory factors was inhibited (Figure 7C). For

381

non-cell death condition, did RIPK3 and MLKL still act effect? As shown in Figure 7B, only

382

RIPK1 and NF-κB inhibition decreased the chemokines release. It can therefore be assumed that

383

RIPK1 inhibitor plays a suppressive role via NF-κB pathway in the absent of cell death. We next

384

detected the relative protein expression in this process. The result suggested that the degradation

385

of IκBα was blocked by RIPK1 inhibitor and it did not involve in the activation of necrosome

386

(Figure 7D). We compared the P65 nuclear translocation between TNF and TSZ treatment in IECs.

387

An early transient intense nuclear import of p65 was observed at 15 and 30 min after TNF

388

stimulation, and a delayed weak translocation of it occurred at 12h to 24h (Figure 7E). Meanwhile,

389

cells began to experience necroptosis process at 3h, and only a brief weak nuclear accumulation

390

was found in cells stimulated by TSZ (Figure 7F). RIPK1 inhibitor could reduce the P65 nuclear

391

translocation mainly occurring in TNF induced IECs injury (Figure 7E and F). This observation

392

may support the conclusion that the suppressive effects of RIPK1 inhibitor on chemokines and

393

adhesion molecules are necroptosis pathway and NF-κB pathway dependent in cell death and

394

non-cell death conditions, respectively.

395

3.7. RIPK1 inhibitor also ameliorated DSS-induced colitis in acute phase

396

In this study, we observed the therapeutic effect of RIPK1 inhibitor on colitis in the recovery

397

phase. To test it in the acute phase, we designed another independent experimental scheme (Figure

398

8A). The results suggested that RIPK1 inhibition ameliorated the progression of the disease: the

399

colon shortening and pathological score were restored. The immunocytes infiltration was reduced

400

in lamina propria and immune organ activation also was suppressed (Figure 8B-F). Therefore, the

401

RIPK1 inhibitor has a protective effect both on acute and recovery phase of colitis via the

402

aforesaid mechanism.

403

3.8. RIPK1 inhibitor alleviated the vicious circle which triggered IECs-immunocytes crosstalk

404

by targeting necroptosis and NF-κB pathway

405

We concluded that RIPK1 inhibitor could suppress inflammation in colitis by maintaining the

406

homeostasis of intestinal barrier, which mainly involves in keeping barrier intact and reducing 16

407

immunocytes infiltration in tissue sites. For one side, RIPK1 inhibitor plays a protective role in

408

IECs via cell death inhibition. For another, it could ameliorate tight junctions’ injury and oxidative

409

stress. Subsequently, damaged IECs could release chemokines and adhesion molecules to recruit

410

immunocytes. RIPK1 inhibitor reduces the production of these molecules from IECs by

411

weakening NF-κB activation. Meanwhile, the DAMPs from IECs initiates immune responses.

412

Subsequently, cytokines released from activated immune cells result in progressive injury of IECs.

413

This process further damages the barrier in a vicious circle, finally leading to colitis. RIPK1

414

inhibitor could suppress the inflammatory response by restraining this positive feedback process

415

(figure9).

416

17

417

4.

Discussion

418

Several researches directed at discovering the proinflammatory function of RIPK1 in

419

tissue-injury diseases which closely related to barrier dysfunction, for instance intestinal

420

inflammation, skin inflammation and renal injury diseases [5, 31, 32]. In this study, an animal

421

model of colitis, which is featured with intestinal mucosal barrier injury, was built to evaluate the

422

effect of RIPK1 inhibitor on intestinal mucosal barrier injury and find the possible mechanisms in

423

crosstalk between epithelium and immune microenvironments. To consider the adverse events of

424

RIPK1 inhibitor and the optimum dose in terms of body weight, we performed a dose dependent

425

study and added a group that normal mice treatment with this inhibitor. The results indicated that

426

the inhibitor had no obvious adverse effects in normal mice (figure1 B-E), and we chose 20mg/kg

427

as the optimum dose for further study (data not shown).

428

The results suggested that RIPK1 inhibitor could ameliorate DSS-induced colitis and

429

suppress the proinflammatory cytokines production (Figure 1). It is worth mentioning that the

430

systematic immune response was weakened under RIPK1i treatment (Figure 2). Nevertheless,

431

very little was found on the question that whether RIPK1 inhibitor has direct suppressive effects

432

on immunocytes.

433

lymphocytes, BMDM and BMDC, to observing whether RIPK1 inhibitor interfered the immune

434

response. As shown in figure3, under antigen stimulation, the immune response was not

435

suppressed by RIPK1 inhibitor. It can therefore be assumed that there were no obvious effects of

436

RIPK1 inhibitor on immunocytes. We would like to focus on the IECs and following events about

437

IECs-immunocytes crosstalk.

To determine it, we simply selected some classic immunocytes, such as spleen

438

Since the immunocytes infiltration in the lamina propria was reduced by RIPK1i inhibitor

439

administration (figure 4A-E), it may play a protective role in the early stages of the disease

440

development. For mucosal barrier integrity, IECs are indispensable in maintaining barrier

441

homeostasis. Damaged IECs contribute to barrier disruption, allowing pathogens and

442

environmental microbes to invade the tissue. The results verified the protective role of RIPK1

443

inhibitor in TSZ induced necroptosis of IECs (figure 5A). On account of the barrier integrity not

444

only depends on IECs but also on the capacity to keep the barrier sealed, it will be necessary to

445

determine whether RIPK1 regulates barrier-associated cell behaviors and characteristics such as 18

446

adhesion[3]. Tight junctions are important components ensuring the integrity and function of the

447

gut epithelial barrier by tightly sealing the intercellular junctions[33]. Our study adds to the

448

evidence that RIPK1 inhibitor could ameliorate the tight junctions’ injury (figure 5C). Intestinal

449

permeability is an important feature of barrier function[34]. And TEER values indicate changes in

450

the cellular monolayer integrity. RIPK1 inhibition displayed positive effects on these indicators

451

(figure 5D and E), which was supported by in vivo study in DSS-induced colitis model. RIPK1

452

inhibitor treatment alleviated barrier destruction during colitis (figure 4F-H). Hence, it could

453

conceivably be assumed that the RIPK1 inhibition ensures intestinal barrier homeostasis by

454

reducing epithelial monolayers disruption and maintaining epithelial permeability. As for the

455

mechanism of RIPK1 inhibitor has a protective role in epithelial barrier injury, we prefer to

456

explore MLCK recruitment and oxidative stress in the following study. Noteworthily, we found

457

that RIPK1 inhibitor could regulate the level of total and cleaved caspase 8 both in colitis and

458

necrotic IECs (data not shown). According to the positive role of caspase 8 in maintaining barrier

459

homeostasis, it is valuable to focus on the mutual regulation of RIPK1 and caspase 8. It may bring

460

enlightenment to the study of cell death-associated diseases. [35].

461

Immunocytes adhesion and migration are critical steps when trafficking in intestine under

462

inflammation status[36]. We consider that RIPK1 plays a key role in the crosstalk between IECs

463

and immunocytes, which may be the pathogenic mechanism of colitis. ICAM-1, a key intercellular

464

adhesion molecule, was reported that it contributed to lymphocytes adhesion and migration across

465

endothelial cells monolayers[37]. There was a large increase of ICAM-1 during TNF stimulation

466

in HT-29 cells, and RIPK1 inhibitor had steady inhibitory effects on the expression level of it

467

(Figure 6A and C). Accordingly, the adhesion ability of IECs and its sticking ability to immune

468

cells were also restrained by RIPK1 inhibitor (figure 6D). Chemokines released by IECs are an

469

initial pathological process for immune cells migration to the focus of inflammation, and it could

470

amplify the destructive immune-inflammatory response in colitis[38]. In the same way, RIPK1

471

inhibition decreased the chemokines production from damaged cells and the chemotaxis process

472

of immunocytes to epithelial cells was also intervened (figure 6E and F). In vivo, the level of

473

ICAM1 and chemokines was increased in colitis model, consistently, the infiltration of

474

immunocytes in intestinal lamina propria was enhanced. According with in vitro data, RIPK1 19

475

inhibitor treatment ameliorated these pathological changes (figure 4I and J). The results

476

demonstrate that RIPK1 has significant effects on epithelial cells to release adhesion molecules

477

and chemokines. So that the following adhesion and migration behaviors of immunocytes and the

478

cascade of epithelial cells disruption events towards immuno-inflammatory responses can be

479

regulated. Our study provides well-reason basis that RIPK1 takes part in the interaction between

480

IECs and immunocytes by promoting adhesion and migration, which should be attached

481

importance to the pathogenesis of injury-associated inflammatory diseases. This mechanism might

482

be a noteworthy point in RIPK1-mediated inflammatory response.

483

To investigate the mechanism of RIPK1-mediated the release of chemokines and adhesion

484

molecules from IECs, we tested which pathway is associated with this effect. Firstly, we found

485

that both under TSZ and TNF stimulation, the cytokines released from IECs could be suppressed

486

by RIPK1 inhibitor (figure 7A). This suggests that RIPK1 inhibitor plays a role in

487

anti-inflammation response both in the presence and absence of cell death. In the cell death

488

process, RIPK1 inhibitor restrained the activation of necrosome, which is an important factor of

489

the inflammatory mediators’ release (figure 7C). Under non-cell death status, RIPK1 inhibitor

490

acted to decrease these cytokines mainly via NF-κB inhibition (figure 7D and E). This observation

491

may support the hypothesis that the ubiquitination and phosphorylation of RIPK1 have mutual

492

effects and this inhibitor plays an indispensable role during these processes. Further research

493

should be undertaken to investigate the involvement of deep molecular mechanisms.

494

Taken together, our findings demonstrate pharmacological mechanisms of RIPK1 inhibitor

495

for treatment colitis. We consider that RIPK1 inhibitor could maintain the homeostasis of barrier

496

and regulate the crosstalk between IECs and lymphocytes, which are not limited to its role in

497

protecting cell death. As a potential anti-inflammation target, RIPK1 has tricky effects in human

498

disease. Therefore, the pharmacological mechanisms of RIPK1 inhibitor in inflammatory diseases

499

urgently need to be clarified. We hope our study could be conducive to a clear understanding of

500

this target and provide new ideas for the treatment of colitis.

501

Correspondence

502

Wei Tang and Jianping Zuo, Shanghai Institute of Materia Medica, Chinese Academy of

503

Sciences, Shanghai 201203, China. E-mail address: [email protected] and [email protected] 20

504 505 506

Conflict of interest The authors declare that they have no competing interests.

Acknowledgements

507

This work was funded by the "Personalized Medicines—Molecular Signature-based Drug

508

Discovery and Development", Strategic Priority Research Program of the Chinese Academy of

509

Sciences (Grant No. XDA12020370) and the Science & Technology Commission of Shanghai

510

Municipality, China (Grant No.18431907100).

511

Author Contributions

512

Huimin Lu performed the in vitro and in vivo experiments, interpreted the data, and wrote the

513

manuscript. Heng Li and Yuxi Yan performed the in vitro experiments and reviewed the

514

manuscript. Chen Fan, Qing Qi, Yanwei Wu, Chunlan Feng, Bing Wu and Yuanzhuo Gao

515

performed the in vivo experiments and provided advice on experimental design. Jianping Zuo

516

provided advice on the experiments and reviewed the manuscript. Wei Tang conceived and

517

supervised the project, designed the experiments, and wrote the manuscript. All authors reviewed

518

the manuscript.

519

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28

653 654 655

Figure legends

656

(A) Experimental design of the DSS-induced colitis. 3% DSS was dissolved in sterile water for 6

657

days except normal control and normal+RIPK1i groups, then the mice were administered with

658

regular water for 3 days. Normal control (HPMC), Normal+RIPK1i (GSK2982772, 20 mg/kg),

659

Vehicle control (HPMC) and RIPK1i (GSK2982772, 20 mg/kg) were given by intragastric

660

administration from day 1 to 9, respectively. During the treatment, weight loss and disease activity

661

index (DAI) were monitored from day 0 to day 9. Ten mice of each group were sacrificed at day

662

10 and the colons, spleens and mesenteric lymph nodes were isolated for subsequent analysis.

663

(B) Body weight (left) and disease activity index (DAI) (right) of 3% DSS treated mice were

664

evaluated (n=10).

665

(C) The typical colon appearance (left) and the statistical analysis length of the colon (right)

666

(n=10).

667

(D) The level of serum ALP and ALT was tested (n=10).

668

(E) Representative microscopic pictures of H&E-stained colons are shown on the left (scale bars,

669

100 μm) and histopathological scores of colonic sections of each group are shown on the right,

670

(n=10).

671

(F) Western blot analysis of representative colon tissues of normal control, vehicle and RIPK1i

672

(20mg/kg) to test the phosphorylation level of RIPK1 (n=4).

673

(G) Pro-inflammatory cytokines of mice serum and colonic tissue were measured by quantitative

674

real-time PCR or ELISA (n=4).

675

(H) The expression of HMGB-1 and HSP90 in colonic tissue was determined through

676

immunohistochemistry (scale bars, 100 μm).

677

Data are presented as means ± SEM. * P < 0.05, ** P < 0.01.

678

Figure.2. RIPK1 inhibitor suppressed the immune response in colitis.

679

(A) The CD4+T cells activation marker CD25, CD69, CD278 were analyzed by flow cytometry in

680

spleen.

681

(B) The percentage of γδT cells in spleen was determined by flow cytometry.

682

(C) The subset of T cells, Th17 differentiation ratio of spleen. This cellular population was gated

Figure.1. RIPK1 inhibitor ameliorated DSS-induced proinflammatory cytokines and DAMPs production.

29

colitis

and

suppressed

the

683

from CD3+ and CD4+.

684

(D) The percentage of macrophage (CD11b+ F4/80+) and neutrophil (CD11b+ly6G+/ly6C+) in

685

spleen and mesenteric lymph nodes.

686

Figure.3. RIPK1 inhibitor has little direct suppressive effects on immunocytes in vitro.

687

(A) Cell counting kit (CCK8) analysis of lymphocytes viability isolated from spleen.

688

(B) Lymphocytes were stimulated with ConA or LPS in the presence or absence RIPK1 inhibitor

689

(GSK2982772). Analysis of cell specific proliferation under antigen treatment by H3-TdR

690

incorporation assay.

691

BMDM and BMDC were pre-incubated with RIPK1i (GSK2982772, 100nM) for 30min and then

692

stimulated with LPS (10μg/ml) for 24h.

693

(C) qPCR analysis of the pro-inflammatory factors in BMDM under LPS stimulation.

694

(D) qPCR analysis of the pro-inflammatory factors in BMDC under LPS stimulation.

695

Figure.4. RIPK1 inhibitor restrained the immunocytes infiltration by maintaining intestinal

696

barrier homeostasis and chemotaxis process in colitis

697

(A) The infiltration of CD3 positive lymphocytes in colon was reflected by immunohistochemistry

698

(scale bars, 100 μm).

699

(B) Flow cytometry analysis the percentage of Treg (CD25+Foxp3+) and Th17 (IL-17+) in

700

lamina propria infiltration. These cellular population were gated from CD3+ and CD4+.

701

(C) MPO activity in the colonic homogenates (n=5).

702

(D) The infiltration of CD11b positive monocytes in colon was determined through

703

immunofluorescence (scale bars, 100 μm).

704

(E) Flow cytometry analysis the percentage of macrophage (CD11b+F4/80+) and neutrophil

705

(CD11b+ly6G+) in lamina propria infiltration.

706

(F) Identification of intestinal epithelial cell injury by TUNEL assay (red) (scale bars, 100 μm).

707

(G) Western blot analysis of representative colon tissues of normal control, vehicle and RIPK1i

708

(20mg/kg) to test the expression level of tight junctions (n=4).

709

(H) Intestinal permeability assay in normal control, vehicle and RIPK1i treatment (20mg/kg)

710

using FITC-labelled dextran, fluorescence intensity in serum was test by microplate reader (n=3).

711

In vivo imaging system was used to reflect the intestinal absorption of fluorescence. 30

712

(I) Western blot analysis of representative colon tissues to test the expression level of adhesion

713

molecule ICAM1 (n=4).

714

(J) Chemokines and receptors of colonic tissue were measured by qPCR (n=4).

715 Data are presented as means ± SEM. * P < 0.05, ** P < 0.01. 716

Figure.5.

717

phosphorylation and accompanying oxidative stress.

718

HT-29 cells or Caco-2 cells (C-D) were pre-incubated with RIPK1i (50nM) or SM164 (50nM) and

719

Z-VAD (25μM) for 30 mins and then culture for different times in the presence or absence of

720

hTNF (100ng/ml).

721

(A) The cell viability was determined by CellTiterGlo after indicated times of TSZ treatment. And

722

TUNEL assay was used to reflect the death of HT-29 cells, damaged of intestinal epithelial cells

723

were stained with red (scale bars, 100 μm).

724

(B) The cell viability was determined by CellTiterGlo after indicated times of TNF treatment.

725

(C) Confocal laser-scanning microscope images of tight junctions E-cadherin, ZO-1 and occludin

726

were measured by immunofluorescence staining (scale bars, 100 μm).

727

(D-E) After TNF administration for 24h, permeability of 4-KDa FITC-dextran (D) and (E)

728

transepithelial electrical resistance (TEER) of Caco-2 cells were determined.

729

(F) After HT-29 cells were stimulated with TNF, cells were harvested respectively at different

730

times for western blot analysis to test the expression of p-MLC.

731

(G) Reactive oxygen species (ROS) production of HT-29 cells cultured with above system was

732

detected by flowcytometry. N-acetyl-cysteine (NAC) is a ROS scavenger, which was used as a

733

positive control.

734

Data are presented as means ± SEM. * P < 0.05, ** P < 0.01.

735

Figure.6. RIPK1 inhibitor could influence the interaction between IECs and immunocytes

736

by suppressing the production of chemokines and adhesion molecules of epithelium.

737

HT-29 cells were pre-incubated with RIPK1i (50nM) for 30 mins and then culture for 24 h in

738

presence or absence of hTNF (100ng/ml) (A-F).

739

(A) qPCR analysis of chemokines on RNA extracted from HT-29 cells.

740

(B) The secretion level of IL-8 and CXCL10 was measured via ELISA.

RIPK1

inhibitor

maintained

IECs

31

homeostasis

by

alleviating

MLC

741

(C) Western blot analysis of adhesion molecule (ICAM-1) in HT-29 cells at different time points

742

after hTNF (50 or 100ng/ml) administration.

743

(D) Representative images of THP-1 cells adhesion to the HT-29 monolayer which treated with

744

the above culture system (scale bars, 100 μm).

745

(E) Representative images of THP-1 cells and Jurkat cells chemotaxis to the conditional

746

supernatant which collected from HT-29 cells treated with the above culture system (scale bars,

747

100 μm).

748

(F) The number of THP-1 and Jurkat cells that were chemotactic to lower chamber was counted

749

by cytometry.

750

Data are presented as means ± SEM of three independent experiments. * P < 0.05, ** P < 0.01.

751

Figure.7. The suppressive effect of RIPK1 inhibitor on cytokines of IECs was related to

752

necroptosis and NF-κB pathway.

753

HT-29 cells were pre-incubated with RIPK1i (50nM) or SM164 (50nM) and Z-VAD (25μM) for

754

30 mins and then culture for different times in presence or absence of hTNF (100ng/ml).

755

(A) qPCR analysis of cytokines on RNA extracted from HT-29 cells at different times after TNF

756

or TSZ treatment.

757

(B) Under necroptosis and NF-κB pathway inhibitors administration (RIPK3i: GSK872 1μM;

758

MLKLi: NSA 1μM; NF-κBi: TPCA-1 1μM), the genes expression of chemokines and adhesion

759

molecule was determined by qPCR with TNF treatment for 24h.

760

(C) Western blot analysis of the protein expression of NF-κB and necroptosis pathway in HT-29

761

cells at different time points after TSZ administration.

762

(D) Western blot analysis of the protein expression of NF-κB and necroptosis pathway in HT-29

763

cells at different time points after TNF administration.

764

(E) Immunostaining of p65 in HT-29 cells treated with TNF in the presence or absence RIPK1i

765

for the indicated time periods (scale bars, 25 μm).

766

(F) Immunostaining of p65 in HT-29 cells treated with TSZ in the presence or absence RIPK1i

767

for the indicated time periods (scale bars, 25 μm).

768

Figure.8. RIPK1 inhibitor also ameliorated DSS-induced colitis in acute phase.

769

(A) Experimental design of the DSS-induced colitis. DSS was dissolved in sterile water for 6 days 32

770

except normal control. Vehicle control (HPMC), RIPK1i (20 mg/kg), were given by intragastric

771

administration from day 1 to 6, respectively. Eight mice of each group were sacrificed at day 6 and

772

the colons, spleens and mesenteric lymph nodes were isolated for subsequent analysis.

773

(B) Body weight (above) and disease activity index (DAI) (below) of 3% DSS treated mice were

774

evaluated (n=6).

775

(C) The typical colon appearance (left) and the statistical analysis length of colon (left) (n=6).

776

(D) Representative microscopic pictures of H&E-stained colons are shown on the left (scale bars,

777

100 μm) and histopathological scores of colonic sections of each group are shown on the right

778

(n=6).

779

(E) The infiltration of γδT cells and monocytes (CD11b+) in lamina propria and its subset

780

percentage of macrophage (CD11b+F4/80+) and neutrophil (CD11b+ly6C+) was analysis by flow

781

cytometry.

782

(F) The CD4+T cells activation markers CD25, CD69, CD278 in spleen and the percentage of

783

macrophage (CD11b+F4/80+) and neutrophil (CD11b+ly6C+) in spleen and mesenteric lymph

784

nodes were analyzed by flow cytometry.

785

(G) Data are presented as means ± SEM. * P < 0.05, ** P < 0.01.

786

Figure.9. RIPK1 inhibitor alleviated the vicious circle which trigger IECs-immunocytes

787

crosstalk by targeting necroptosis and NF-κB pathway

788

RIPK1 inhibition ameliorates colitis at the early stage during disease progress via protecting

789

epithelial barrier injury and regulating IECs-immunocytes crosstalk.

790

Author Contributions

791

Huimin Lu performed the in vitro and in vivo experiments, interpreted the data, and wrote the

792

manuscript. Heng Li and Yuxi Yan performed the in vitro experiments and reviewed the

793

manuscript. Chen Fan, Qing Qi, Yanwei Wu, Chunlan Feng, Bing Wu and Yuanzhuo Gao

794

performed the in vivo experiments and provided advice on experimental design. Jianping Zuo

795

provided advice on the experiments and reviewed the manuscript. Wei Tang conceived and

796

supervised the project, designed the experiments, and wrote the manuscript. All authors reviewed

797

the manuscript.

798 33

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