Elsevier

Human Immunology

Volume 69, Issue 12, December 2008, Pages 845-850
Human Immunology

Commensal intestinal bacterial strains trigger ankylosing enthesopathy of the ankle in inbred B10.BR (H-2k) male mice

https://doi.org/10.1016/j.humimm.2008.08.296Get rights and content

Abstract

Joint disease ankylosing enthesopathy (ANKENT) naturally occurs in inbred mice with C57Bl/10 genetic background. ANKENT has many parallels to human ankylosing spondylitis (AS) and represents an animal model for AS. Environmental conditions (i.e., microbial load of the organism) are among the risk factors for ANKENT, similar to AS. The role of microflora in the development of ANKENT was investigated. ANKENT was tested in four experimental groups of germ-free mice associated with different numbers of various intestinal microbes and three control groups: germ-free, specific pathogen-free, and conventional (CV) mice. Mice were colonized either with anaerobic bacteria isolated from the intestine of a CV mouse or with bacterial strains obtained from the collection of microorganisms. Microbes were characterized and checked by microbiological cultivation methods and with the use of polymerase chain reaction amplification and rDNA sequence analysis. Joint disease developed in GF mice colonized with a mixture containing Bacteroides spp. and Enterococcus sp., and/or Veillonella sp. and Staphylococcus sp. No ANKENT appeared in males colonized with probiotic bacterium Lactobacillus sp. In control groups ANKENT occurred in SPF and CV animals; the GF animals remained healthy. The results confirmed that the germ-free conditions protect from joint inflammation, and thus microbes are necessary for ANKENT development. In colonized mice the ANKENT was triggered by luminal anaerobic bacteria, which are common components of intestinal microflora.

Introduction

Spondyloarthropathies (SpA) are a group of inflammatory disorders with 0.3% prevalence in the adult human population. Major afflicted target organs include joints and enthesis, with the latter defined as the area of insertion of ligaments, tendons, or joint capsules into bone. Enthesitis is a highly specific mark of SpA [1], [2]. Among spondyloarthropathies, ankylosing spondylitis (AS) has been more intensively studied for its strong association with the major histocompatibility antigen HLA-B27 [3], [4] and various animal models have been established for this purpose [5], [6], [7], [8], [9], [10]. Our animal model relies on the observation that joint disease ankylosing enthesopathy (ANKENT) occurs in certain mouse strains with multiple features similar to those found in AS in humans.

Joint disorder ANKENT was first observed in transgenic mice with two human genes, major histocompatibility gene class I HLA-B27 and β2-microglobulin. The screening of B27 transgenic lines revealed an enthesitis of the ankle and tarsal joints in the hind paws of some male mice [6]. A similar observation was later noted by screening inbred mouse strains maintained in animal houses in the Institute of Molecular Genetics in Prague and the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service in Amsterdam [11], [12]. The ANKENT occurred naturally in males of B10.BR and other H-2 congenic strains with a C57BL/10 (B10) genetic background. Thus, the disease was not caused only by the effects of transgene expression. However, an association of ANKENT with the human HLA-B27 gene was obvious: in B27 transgenic animals the prevalence of ANKENT was significantly higher [12].

ANKENT resembles AS both in factors influencing the disease development and in the histopathology of affected tissues. The disease begins with a slight infiltration of inflammatory cells at the site of the enthesis, followed by the proliferation of cartilage and connective tissue, and culminates in ankylosis of the ankle and tarsal joints [13]. Risk factors in AS include gender (prevalence in males), age (first symptoms usually seen in young adults), and association with HLA-B27. ANKENT incidence also depends on the gender and age, as well as on genetic factors [12], [13], [14].

Relevant risk factors for both SpA and ANKENT include environmental conditions. Evidence exists that SpA, especially reactive arthritis, manifested itself after bacterial infection [15], [16]. Results in germ-free (GF), specific pathogen-free (SPF), and conventional (CV) mice documented an effect of microbial load on the ANKENT incidence. SPF males developed ANKENT at a lower frequency than their conventional counterparts [12] and ANKENT did not occur in B10.BR germ-free males, whereas their conventionalized littermates developed the joint disease [17]. Another reported environmental risk factor for ANKENT is grouped caging of male mice [18].

Similar findings were reported in rats. In conventional transgenic rat lines carrying human β2-microglobulin and high copy numbers of HLA-B27, gut and joint inflammations were prominent aspects of spontaneous multisystem disorders [7]. When these transgenic rats were reared under germ-free conditions, they did not develop inflammatory intestinal or peripheral joint disease [19]. Colonization of germ-free B27 transgenic rats with normal luminal bacteria induced gastrointestinal and systemic inflammation with joint involvement, and the participation of microorganisms in this process was clearly demonstrated [20], [21].

In this paper we focused on the effect of indigenous microorganisms derived from intestinal microflora in mouse ANKENT development. We tested the capability of intestinal microflora to induce activation of the immune system resulting in joint disorder manifestation.

Section snippets

Animals

Males of inbred mouse strain B10.BR (C57Bl/10 genetic background, H-2k haplotype), which is ANKENT prone, were used. Mice were placed four to six per cage and the incidence of ANKENT was recorded in the following categories: SPF mice housed in SPF conditions (the breeding pairs of SPF mice were provided by the IMG Production Unit, Prague), CV mice housed in CV facilities, and GF mice and GF mice associated with bacteria (gnotobionts) housed in sterile Trexler-type plastic isolators. Details are

ANKENT occurrence

Development of joint disease ANKENT was long-term controlled (until 12 months of age) in four experimental groups of gnotobiotic animals that were obtained after the association of germ-free mice with various numbers of different bacterial strains (mono-, di-, and polyassociation; groups B, C, D, and E). Simultaneously, GF, SPF, and CV mice were used as controls (groups A, F, and G). A radiograph of the afflicted joint of an ANKENT-positive male is illustrated in Figure 1 and the findings are

Discussion

Several animal models have been established to investigate the causes of human spondyloarthopathies [10]. A significant role in disease development, beside the genetic factors, gender and age, is played by the environmental conditions in which the animals are maintained. Almost all manifestations of multisystemic disorder disappeared in HLA-B27 transgenic rats reared under germ-free conditions [19]. Nontransgenic mice susceptible to ANKENT did not develop the disease at all in the absence of

Acknowledgments

We thank Prof. H. Tlaskalova and Dr. S. Takacova for critical reading and advice regarding the preparation of the manuscript. This work was supported by Grant 305/03/0287 from the Grant Agency of the Czech Republic, Grant 2B08028 from the Ministry of Education, Youth and Sports, and the Institutional Research Concept Funds of the Academy of Sciences of the Czech Republic AVOZ50520701.

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    This study is dedicated to Prof. Pavol Ivanyi, MD, PhD.

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