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A Proteome Analysis of Rheumatoid Arthritis Gut Mucosa Tue Bjerg Bennike, Torkell Ellingsen, Henning Glerup, Ole Kristian Bonderup, Thomas Gelsing Carlsen, Michael Kruse Kruse Meyer, Martin Bøgsted, Gunna Christiansen, Svend Birkelund, Vibeke Andersen, and Allan Stensballe J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00598 • Publication Date (Web): 14 Sep 2016 Downloaded from http://pubs.acs.org on September 15, 2016
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A Proteome Analysis of Rheumatoid Arthritis Gut Mucosa Tue Bjerg Bennike1,*, Torkell Ellingsen2,3, Henning Glerup3,4, Ole Kristian Bonderup3,4, Thomas Gelsing Carlsen5, Michael Kruse Meyer5,6, Martin Bøgsted7,8, Gunna Christiansen9, Svend Birkelund5, Vibeke Andersen10,11,‡, Allan Stensballe5,‡
1 Departments of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, United States 2 Department of Rheumatology, Odense University Hospital, Odense DK-5000, Denmark 3 University Research Clinic for Innovative Patient Pathways, Aarhus University, Aarhus DK8000, Denmark 4 Diagnostic Center, Section of Gastroenterology, Regional Hospital Silkeborg, Silkeborg DK8600, Denmark 5 Department of Health Science and Technology, Aalborg University, Aalborg DK-9220, Denmark 6 Department of Rheumatology, and Center for Clinical Research, North Denmark Regional Hospital, Hjoerring DK-9800, Denmark
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7 Department of Clinical Medicine, Aalborg University, Aalborg DK-9220, Denmark 8 Department of Haematology, Aalborg University Hospital, Aalborg DK-9000, Denmark 9 Department of Biomedicine, Aarhus University, Aarhus DK-8000, Denmark 10 Institute of Regional Health Research-Center Soenderjylland, University of Southern Denmark, Odense DK-5230, Denmark 11 Molecular Diagnostic and Clinical Research Unit, Hospital of Southern Jutland, Aabenraa DK-6200, Denmark
KEYWORDS Rheumatoid arthritis; dihydrofolate reductase; DHFR; methotrexate; leflunomide; citrullination; proteomics; anti-citrullinated protein antibody; ACPA; colon mucosa
ABSTRACT Rheumatoid arthritis (RA) is an inflammatory joint disease leading to cartilage damage and ultimately impaired joint function. To gain new insight into the systemic immune manifestations of RA, we characterized the colon mucosa proteome from 11 RA-patients and 10 healthy controls. The biopsies were extracted by colonoscopy and analyzed by label-free quantitative proteomics, enabling the quantitation of 5,366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared to controls, and correlated with the administered dosage of methotrexate (MTX), the most frequently prescribed immunosuppressive drug for RA. Additionally, our data suggests that treatment with Leflunomide, a common alternative to MTX, increases DHFR. The findings were
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supported by immunohistochemistry with confocal microscopy, which furthermore demonstrated that DHFR was located in the cytosol of the intestinal epithelial- and interstitial cells. Finally, we identified 223 citrullinated peptides from 121 proteins. Three of the peptides were unique to RA. The list of citrullinated proteins was enriched in extracellular- and membrane proteins, and included known targets of anti-citrullinated protein antibodies (ACPAs). Our findings support that the colon mucosa could trigger the production of ACPAs, which could contribute to the onset of RA. The MS-data has been deposited to ProteomeXchange with identifiers PXD001608 and PXD003082.
INTRODUCTION Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, affecting 0.5 to 1.0% of adults in developed countries.1 The first-line treatment of RA consists of disease-modifying antirheumatic drugs (DMARDs), which inhibits the progression of RA.2 A low dosage of methotrexate (MTX) (15-25 mg/week) is the most frequently prescribed DMARD for RA, and is also used in the treatment of other inflammatory diseases.3 Up to 71% of RA-patients are positive for serum autoantibodies targeting citrullinated proteins (ACPAs), which is associated with a more aggressive disease, bone erosion, and increased mortality.4 Many indications have been found that ACPA-positive RA is caused by a break of immune tolerance to citrullinated proteins.4–9 The ACPAs can commonly be measured prior to onset of clinical inflammation in the joints, suggesting that the initial site of disease triggering could take place elsewhere.10 Suggested sites include the mucosal surfaces where the immune system is in direct contact with the environment, e.g. in lung- and gut tissue.11,12 Smoking has been found to increase
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citrullination in the lung tissue, smoking is the best known risk factor for RA, and a recent study has found two citrullinated vimentin peptides shared between the synovial fluid and lung tissue from RA-patients.12–15 Changes to the gut microbiota have also been linked to RA, where an over-representation of gram-positive bacteria and an under-representation of gram-negative bacteria have been described.16 Most studies on the gut and RA are, however, mainly based on animal studies, and several recent publications have concluded that more research on RA and the gut in humans is needed.5,17,18 Given the systemic nature of RA and the link to phylogenetic changes in the gut microbiota, we hypothesized that sub-clinical intestinal inflammation may be involved in RA, where the colon mucosa proteome of gastroenterological healthy RA-patients remains uninvestigated. In this study, we therefore characterized the proteome of the colon mucosa of RA-patients in comparison to healthy controls, with an emphasis on citrullinated peptides and proteins.
EXPERIMENTAL PROCEDURES Collection of Colon Mucosa Biopsies At The Diagnostic Center, Regional Hospital Silkeborg, Denmark, 11 RA-patients without known gastrointestinal disease and 10 healthy controls were recruited in the period from 2012 to 2013 for the project. Oral and written informed consent was obtained from all participants prior to participation in the study, and the project was approved by The Regional Scientific Ethical Committee (S-20120204) and the Danish Data Protection Agency (2008-58-035). Colon mucosa biopsies were sampled 40 cm from the anus by sigmoidoscopy, and diagnosis, medication (Table
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1), and prognostic data (Table 2) were recorded from the patient records. Biopsies for mass spectrometry (MS) were immediately transferred to cryotubes and snap-frozen in liquid nitrogen followed by storage at -80°C until proteomics sample preparation. Biopsies
for
immunohistochemistry were immediately placed on mixed cellulose ester filters (Advantec, Tokyo, Japan), fixed in phosphate buffered 4% formaldehyde, and stored at room temperature for 24 hrs. before paraffin embedding according to standard pathology procedures. The biopsies were obtained and processed in parallel with a study of ulcerative colitis, and the control samples were used in both studies.19
Table 1. Participant data and medical information. ID
Age Gender Medication MTX LFM Other [mg/w] [mg/d]
Ctrl_1
54
Male
0
0
-
Ctrl_2
27
Male
0
0
Novorapid 35 [IE/d]
Ctrl_3
42
Male
0
0
-
Ctrl_4
48
Male
0
0
-
Ctrl_5
54
Female 0
0
-
Ctrl_6
27
Female 0
0
-
Ctrl_7
42
Female 0
0
-
Ctrl_8
23
Male
0
0
-
Ctrl_9
28
Male
0
0
-
Ctrl_10 31
Male
0
0
-
RA_1
62
Male
15
0
Plaq 200, [mg/d] salaz 3000 [mg/d]
RA_2
47
Male
0
10
Simponi 50 [mg/month], 3000 salaz [mg/d]
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RA_3
60
Female 25
0
Plaq 200 [mg/d]
RA_4
59
Female 0
20
-
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FA 10 [mg/w], Centyl 575 [mg/day], RA_5
59
Male
25
0
RA_6
55
Female 15
0
Alendronate 70 [mg/w], Pantoprazol 40 [mg/d] Salaz 2000 [mg/d], plaq 200 [mg/d] certolizumab 200 [mg/2w]
RA_7
60
Female 17.5
0
Adalimumab 80 [mg/w], FA 5 [mg/w]
RA_8
59
Female 15
0
Plaq 200 [mg/d], salaz 2000 [mg/d] Plaq 200 [mg/d], salaz 1000 [mg/d],
RA_9
70
Female 15
0
FA 10 [mg/w], simv 40 [mg/d], acetylsalicylic acid, metoprolol, amlodipine
RA_10 69
Female 15
0
Simv 40 [mg/d], FA 10 [mg/w]
RA_11 57
Male
0
Simv 40 [mg/d], FA 5 [mg/w]
25
MTX: methotrexate, LFM: leflunomid, DHFR: dihydrofolate reductase, FA: folic acid, salaz: salazopyrin, plaq: plaquenil, simv: simvastatin.
Table 2. Rheumatoid arthritis (RA) clinical information. ID
Measure
VAS
Assessment TJ
SJ
HAQ
CRP Anti- IgMDAS28 Pain [mg/L] CCP RF
Fatigue
RA_1
