Targeting a Cysteine Protease from a Pathobiont Alleviates Experimental

Peng et al. Arthritis Research & Therapy (2020) 22:114 https://doi.org/10.1186/s13075-020-02205-z RESEARCH ARTICLE Open Access Targeting a cysteine protease from a pathobiont alleviates experimental arthritis Hsin-Yi Peng1†, Shih-Yao Chen2†, Shih-Hong Siao3, Jinghua Tsai Chang4, Ting-Yin Xue1, Yi-Hsuan Lee1, Ming-Shiou Jan6, Gregory J. Tsay1,5*† and Moncef Zouali7,8,9*† Abstract Background: Several lines of evidence suggest that the pathobiont Porphyromonas gingivalis is involved in the development and/or progression of auto-inflammatory diseases. This bacterium produces cysteine proteases, such as gingipain RgpA, endowed with the potential to induce significant bone loss in model systems and in patients. Objective: We sought to gain further insight into the role of this pathobiont in rheumatoid arthritis (RA) and to identify novel therapeutic targets for auto-inflammatory diseases. Methods: We profiled the antibody response to RgPA-specific domains in patient sera. We also tested the potential protective effects of RgpA domains in an experimental arthritis model. Results: Pre-immunization of rats with purified recombinant RgpA domains alleviated arthritis in the joints of the rodents and reduced bone erosion. Using a functional genomics approach at both the mRNA and protein levels, we report that the pre-immunizations reduced arthritis severity by impacting a matrix metalloprotease characteristic of articular injury, a chemokine known to be involved in recruiting inflammatory cells, and three inflammatory cytokines. Finally, we identified an amino acid motif in the RgpA catalytic domain of P. gingivalis that shares sequence homology with type II collagen. Conclusion: We conclude that pre-immunization against gingipain domains can reduce the severity of experimentally induced arthritis. We suggest that targeting gingipain domains by pre-immunization, or, possibly, by small-molecule inhibitors, could reduce the potential of P. gingivalis to translocate to remote tissues and instigate and/or exacerbate pathology in RA, but also in other chronic inflammatory diseases. Keywords: Rheumatoid arthritis, Periodontitis, Porphyromonas gingivalis, Gingipain, Collagen-induced arthritis Key messages Targeting pathobiont could alleviate pathology in chronic inflammatory diseases. Pathobionts can translocate to remote tissues and instigate pathology. Introduction Pre-immunization against a cysteine protease from a Rheumatoid arthritis (RA) and periodontitis (PD) are pathobiont reduces disease severity. two common human chronic inflammatory diseases. RA is the most frequent autoimmune disease, and its eti- * Correspondence: [email protected]; [email protected] †Hsin-Yi Peng, Shih-Yao Chen, Gregory J. Tsay and Moncef Zouali contributed ology remains unknown. With a significant prevalence equally to this work. worldwide, PD is an inflammatory disorder of oral tis- 1 Division of Immunology and Rheumatology, China Medical University sues, including the gingiva, cementum, and periodontal Hospital, Taichung, Taiwan 7Inserm UMR 1132, F-75475 Paris, France ligament. For decades, associations between RA and PD Full list of author information is available at the end of the article have been noted, and mounting evidence suggests that © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Peng et al. Arthritis Research & Therapy (2020) 22:114 Page 2 of 15 PD-associated microbes could be involved in RA patho- an Arg-specific catalytic domain (CD), and a large C- genesis and that the presence of the pathobiont Porphyr- terminal hemagglutinin/adhesin (HD) region (Fig. 1). omonas gingivalis represents a risk in the development In animal models, oral challenge with P. gingivalis was and/or progression of RA [1–3]. First, there is an in- demonstrated to stimulate bone loss [13]. Further inves- creased prevalence of PD in RA patients, as compared to tigation disclosed that gingipains produced by P. gingiva- controls [4, 5]. Second, the presence of antibodies to P. lis have the potential to modulate the host immune gingivalis is associated with that of RA-related autoanti- response by affecting both the innate and adaptive bodies in subjects at increased risk for disease, but who branches of immunity, i.e., by degrading defensins [14], have not yet developed RA [1]. Third, serum antibodies suppressing the cascade of complement activation [15], to P. gingivalis are present in significantly higher con- cleaving molecules expressed on T cells [16], proteolytic- centrations in patients with RA than in healthy controls, ally inactivating both anti-inflammatory (IL-4, IL-5) and and their levels are in correlation with the presence of pro-inflammatory (IL-12, IL-1b, IFN-g, and TNF-a) cyto- anti-citrullinated protein antibodies (ACPAs) that spe- kines [17], and stimulating IL-6 production by oral epi- cify RA disease severity [1, 2]. Fourth, concentrations of thelial cells [18] and IL-8 production by gingival circulating anti-P. gingivalis antibodies are associated fibroblasts [19], thereby hampering the protective im- with the expression of ACPAs detectable in the gingival mune response and enhancing inflammatory responses. crevicular fluid of PD patients [2, 6]. Fifth, citrullinated Experimental studies also revealed that the gingipains antigens that are present in the periodontium of PD pa- Kgp and Rgp exert distinct virulence roles. tients are thought to play a role in triggering the im- Immunization of mice with RgpA could stimulate the mune response observed almost exclusively in RA [7]. production of P. gingivalis- and RgpA-specific IgG anti- Lastly, P. gingivalis DNA was found in the synovial fluid bodies, and the immunized animals against P. gingivalis of patients with RA [8]. Yet the precise mechanisms or purified RgpA, but not against RgpB, were protected underlying the interplay between the pathobiont, RA, from maxillary bone loss [20]. Consistently, mice orally and PD remain to be elucidated. infected with either P. gingivalis wild type or gingipain − − − − − − − − P. gingivalis has evolved a cell surface-associated pro- mutants (RgpA / , Kgp / , RgpA / /Kgp / ) exhibited a teolytic system composed of several unique enzymes. In similar trend, and only RgpA-expressing bacteria showed addition to participating indirectly in the pathological significant bone loss and reduced phagocytosis, resulting destruction of periodontal tissues, these enzymes also in increased survival of P. gingivalis in the chamber exu- allow the bacterium to evade host defense mechanisms dates [21]. Together, the facts (i) that elimination of [9]. Among these virulence factors, cysteine proteases RgpA from P. gingivalis diminishes inflammation and are endowed with a potential to deregulate the host im- augments phagocytosis and antibody titers, coincidental mune response. Notably, P. gingivalis produces three with reduced alveolar bone loss [21], and (ii) that only cysteine proteinases known as gingipains: lysine-specific immunization of animals against purified RgpA, but not gingipain (Kgp), arginine-specific gingipain A (RgpA), against RgpB, affords protection from bone loss [20] and arginine-specific gingipain B (RgpB) [10–12]. They demonstrate that RgpA gingipain acts as a major viru- are essentially present on the outer membranes and lence factor in pathogenesis. outer membrane vesicles of virtually all P. gingivalis Even though experimental evidence indicates that gin- strains. The protein encoded by the rgpa gene is com- gipains represent major virulence factors of P. gingivalis, posed of a signal peptide, an N-terminal pro-fragment, the relative contributions of the catalytic and Fig. 1 Protein domains of the RgpA gingipain complex. The catalytic, arginine-specific adhesin domain (CD) comprises 492 amino acids (positions 228–719). The hemagglutinin domain (positions 720–1703) is composed of 984 amino acids and comprises four sub-domains: HA1 (positions 720–1136), HA2 (positions 1137–1271), HA3 (positions 1272–1429), and HA4 (positions 1430–1703) [29] Peng et al. Arthritis Research & Therapy (2020) 22:114 Page 3 of 15 hemagglutinin domains of RgpA to pathogenesis in P. final concentration of 1 mM for approximately 14 h. gingivalis-mediated human diseases remain unclear. To After induction, the bacteria were centrifuged at 8000g gain further insight into the potential roles of P. gingiva- for 10 min at 4 °C and stored at − 80 °C until use. The lis gingipains in RA and PD, we therefore elected to pellet (2 g) was resuspended in 40 ml of lysis buffer

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