J Proteome Res. 2017 Jan 6;16(1):346-354. doi: 10.1021/acs.jproteome.6b00598.

Proteome Analysis of Rheumatoid Arthritis Gut Mucosa.

Bennike TB1, Ellingsen T2,3, Glerup H3,4, Bonderup OK3,4, Carlsen TG5, Meyer MK5,6, Bøgsted M7,8, Christiansen G9, Birkelund S5, Andersen V10,11, Stensballe A5.

1Departments of Pathology, Boston Children’s Hospital and Harvard Medical School , Boston, Massachusetts 02115, United States.
2Department of Rheumatology, Odense University Hospital , Odense DK-5000, Denmark.
3University Research Clinic for Innovative Patient Pathways, Aarhus University , Aarhus DK- 8000, Denmark.
4Diagnostic Center, Section of Gastroenterology, Regional Hospital Silkeborg , Silkeborg DK-8600, Denmark.
5Department of Health Science and Technology, Aalborg University , Aalborg DK-9220, Denmark.
6Department of Rheumatology and Center for Clinical Research, North Denmark Regional Hospital , Hjoerring DK-9800, Denmark.
7Department of Clinical Medicine, Aalborg University , Aalborg DK-9220, Denmark.
8Department of Haematology, Aalborg University Hospital , Aalborg DK-9000, Denmark.
9Department of Biomedicine, Aarhus University , Aarhus DK-8000, Denmark.
10Institute of Regional Health Research-Center Soenderjylland, University of Southern Denmark , Odense DK-5230, Denmark.
11Molecular Diagnostic and Clinical Research Unit, Hospital of Southern Jutland , Aabenraa DK-6200, Denmark.



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 5366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared with controls and correlated with the administered dosage of methotrexate (MTX), the most frequently prescribed immunosuppressive drug for RA. Additionally, our data suggest that treatment with Leflunomide, a common alternative to MTX, increases DHFR. The findings were 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 anticitrullinated 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 have been deposited to ProteomeXchange with identifiers PXD001608 and PXD003082.


ACPA; DHFR; anticitrullinated protein antibody; citrullination; colon mucosa; dihydrofolate reductase; leflunomide; methotrexate; proteomics; rheumatoid arthritis

PMID: 27627584



For webpage:

The colon mucosa is a potential site for the initial triggering of Rheumatoid Arthritis Journal of Proteome Research, The Immune System and the Proteome 2016 special issue

A study of proteins reveals that the colon mucosa is a potential site of immune tolerance break towards proteins with the post-translational modification citrullination, which could contribute to the onset of the joint disease rheumatoid arthritis.



Rheumatoid arthritis (RA) is a chronic inflammatory disease, affecting 0.5 to 1.0% of adults in industrialized countries. The disease is characterized by a systemic inflammation which mainly affects the joints.[1] The cause for the initial onset of RA remains unknown. However, up to 71% of RA-patients can be tested positive for serum autoantibodies targeting citrullinated proteins (ACPAs), including the proteins vimentin, fibrin, and fibrinogen.[2] The detecting of serum ACPAs is today used as a clinical marker to diagnose RA. As such, many studies points to that RA is caused by a break of immune tolerance toward citrullinated proteins.[2]–[6]

The post-translational modification of proteins is a common biological process and is often crucial to ensure the correct physiological function of the protein. Citrullination is the deimination of protein arginine residues, yielding the non-standard amino acid citrulline (Figure 1). The reaction is mediated by the peptidylarginine deiminase (PAD) family of enzymes. At physiological pH, arginine is positively charged, in contrast to citrulline, which is neutral. In many cases, this altered charge has a major impact on the protein fold and functions, but the exact role of citrullination remains enigmatic. [7]



Figure 1. Peptidylarginine deiminase (PAD) citrullination of arginine residues into citrulline on a protein. At physiological pH arginine is positively charged, in contrast to citrulline which is negative.


The ACPAs can often be measured before inflammation can be detected in the joints in RA. This suggests that the initial site of disease triggering can take place elsewhere.[8] Suggested sites include the mucosal surfaces where the immune system is in direct contact with the environment, e.g. in the gut tissue.[9], [10] Changes to the gut microbiota have been associated with RA.[11] The colon mucosa is the main interacting surface between the gut microbiota and the immune system. However, there is a lack of human studies on the gut and RA, where most currently is based on animal studies.[3], [12], [13] Therefore, we conducted the first study of the proteins (the proteome) in the RA colon mucosa, of 11 RA-patients and 10 healthy controls.[14] We put an emphasis on the study of citrullinated peptides and proteins using a recently published mass spectrometry-based proteomics method and protocols optimized for colon tissue.[15]–[18] The MS data has been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with dataset identifiers PXD001608 and PXD003082.[19], [20]



Figure 2. Representative 2D Chromatogram of a mass spectrometry analysis of a colon biopsy. Identified dihydrofolate reductase (DHFR) peptides indicated. The identified peptides originating from the 5366 identified proteins are plotted from low (white) to high (green) signal intensity.


The global proteomics analysis (Figure 2), identified, for the first time, an increased abundance of the enzyme dihydrofolate reductase (DHFR) in the colon mucosa tissue of RA patients treated with methotrexate (MTX), the most frequently prescribed immunosuppressive drug for RA. Additionally, our data suggest that treatment with Leflunomide (Lef), a common alternative to MTX, also increases DHFR colon mucosa abundances (Figure 3). The compensatory increase of DHFR was found to correlate positively with the administered MTX + Lef dosage. The findings were validated using confocal microscopy which furthermore demonstrated that DHFR was located intracellularly in the intestinal epithelial and interstitial cells (Figure 4).



Figure 3. Protein abundance of dihydrofolate reductase (DHFR) in colon mucosa biopsies from rheumatoid arthritis (RA) patients and controls. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means and gray bars indicate 95% confidence intervals of the means; data points are plotted as open circles, color shows administered methotrexate (MTX) [mg/w] + leflunomid (Lef) [mg/d] dosage.



Figure 4. Immunofluorescence confocal microscopy of colon sections stained for dihydrofolate reductase (DHFR) (green) and DNA (red). The methotrexate (MTX)-treated rheumatoid arthritis (RA) patients (a) show higher staining for DHFR than controls (b). Scale bar: 100 μm.


The citrullination analysis identified 223 citrullinated peptides from 121 different proteins. None of the citrullinated peptides were measured having a statistically significantly changed abundance, between RA patients and controls (q-value < 0.05). However, three peptides were unique to colon mucosal biopsies from RA patients. The majority of the identified citrullinated proteins were known to be exposed to the extracellular environment, which can facilitate directly interactions with the immune system. The list of citrullinated proteins included fibrinogen alpha, vimentin, and actin, which are known targets of ACPAs in RA. Our findings support that the colon mucosa is a potential site of immune tolerance break and the triggering of ACPA production, which could contribute to the onset of RA.



The Lundbeck Foundation (2014-3372), the Carlsberg Foundation (CF14-0561), and the A. P. Moller Foundation Denmark are acknowledged for grants enabling the project (TBB grants). Knud and Edith Eriksens Memorial Foundation, the Lundbeck Foundation (R126-2012-12194) and Ferring are acknowledged for grants, enabling the collection of the biological sample material (VA grant). The Obelske Family Foundation, the SparNord Foundation and the Svend Andersen Foundation are acknowledged for grants supporting the analytical platform (AS grants).



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