Rheumatoid Arthritis is an Autoimmune Disease Triggered by Proteus Urinary Tract Infection

Rheumatoid arthritis (RA) is a chronic and disabling polyarthritic disease, which affects mainly women in middle and old age. Extensive evidence based on the results of various microbial, immunological and molecular studies from different parts of the world, shows that a strong link exists between Proteus mirabilis microbes and RA. We propose that sub-clinical Proteus urinary tract infections are the main triggering factors and that the presence of molecular mimicry and cross-reactivity between these bacteria and RA-targeted tissue antigens assists in the perpetuation of the disease process through production of cytopathic auto-antibodies. Patients with RA especially during the early stages of the disease could benefit from Proteus anti-bacterial measures involving the use of antibiotics, vegetarian diets and high intake of water and fruit juices such as cranberry juice in addition to the currently employed treatments.


Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, which affects millions of people all around the world, with a prevalence rate ranging from 0.5 to 1% (Lawrence et al. 1998). The disease in the majority of patients takes a mild to moderate course, whilst in others it has a more disabling consequence, which might have a great effect on the socio-economic status of the patient (Cooper 2000).
RA affects individuals of middle age groups and occurs three times more frequently in women than in men.

Aetiopathogenesis
A general scientific consensus exists, which considers RA as an immune-mediated disease that could possibly be triggered by an environmental (microbial) factor in a genetically susceptible individual.
Extensive evidence supports the role of cellular and humoral autoimmunity in the development of RA, and some of these are listed as follows: 1. Predominant role of B lymphocytes in the pathogenesis of RA ) and signs of accumulations of immunoglobulins and other inflammatory products such as complements at the site of synovial pathological lesions in RA patients (Low and Moore 2005). 2. Detection of elevated levels of auto-antibodies in the serum and/or synovial fluid of patients with RA (Rantapaa-Dahlqvist 2005). 3. Significant improvements in RA disease parameters following B cell depletion therapy, e.g. with the use of anti-CD20 antibodies (Perosa et al. 2005).

Role of HLA genes in RA
The role of genetics in development of RA has been examined mainly through family, twin and molecular analytical studies. For instance, familial distribution of RA among first-degree relatives (Deighton et al. 1992a) and twins (MacGregor et al. 2000), indicates that RA runs in some families, basically supporting the concept of a genetic contribution, but at the same time arguing against the proposition that RA could be considered as a purely genetic disorder. However, in two separate genome-wide screenings with affected sib pairs, it has been shown that HLA haplotypes had the largest genetic contribution in RA (Cornelis et al. 1998;Jawaheer et al. 2001). Nearly three decades ago, HLA-DR4 was the first genetic marker among other class II molecules, found to be significantly liked with RA susceptibility (Stastny 1976). It has been shown later that various other HLA-DRB1 alleles were either associated (HLA-DRB * 0101, * 0102, * 0401, * 0404, * 0405, * 1001 and 1402) or not associated (HLA-DRB * 0103, * 0402, * 0403 and * 0408) with RA (Newton et al. 2004), based on certain differences occurring within amino acid sequences at the location 69 -74 "EQRRAA" which has been termed as the "shared epitope" (SE) moiety (Gregersen et al. 1987).
Regardless of the difference in the distribution of HLA genes among various ethnic groups, it has been reported that more than 95% of patients possess at least one of the RA-linked HLA-DR molecules (Weyand et al. 1992), which contain the "SE" amino acid sequence. Further to its role in conferring the disease susceptibility, these RA-associated HLA-DRB1 alleles have also been reported to have a great impact on the disease severity and extra-articular manifestations in patients with RA (Turesson et al. 2005).
Immunological and molecular links between RA and Proteus During the last 3 decades an extensive literature on the link between Proteus microbes and RA has been published in peer-reviewed journals. These studies were carried out by various independent groups worldwide. Some of this evidence, listed in chronological orders, can be summarized as follows: (Table I) 1. In early 1970s, a group from Tennessee reported an increase in the geometric mean titres of antibodies to Proteus OXK and herpes virus hominis but not to 28 other infectious disease antigens in RA patients when compared to controls (Chandler et al. 1971). 2. In mid 1980s, our group reported for the first time that antibody levels against P. mirabilis, a urinary pathogen, were significantly elevated in RA patients when compared to those with ankylosing spondylitis (AS) or healthy control subjects (Ebringer et al. 1985). 3. In early 1990s, we have identified an amino acid sequence homology between the "EQ/KRRAA" amino acid motif present in RA HLA-susceptibility molecules and the "ESRRAL" sequence present in P. mirabilis haemolysins (Ebringer et al. 1992).
4. In mid 1990s, our group has found another molecular homology between "LRREI" amino acid sequence present in type XI collagen and "IRRET" amino acid motif present in Proteus urease enzymes . 5. In late 1990s, in vitro and in vivo immunological cross reactivities have been observed between synthetic peptides from Proteus haemolysin and urease enzyme molecules and those of HLA-DR1/DR4 and collagen self-antigens (Tiwana et al. 1999). Furthermore, Senior and colleagues from Dundee, have found that Proteus mirabilis is the most prominent microbe encountered in the urine of RA patients, who had bacteriuria more frequently than age-and sex-matched healthy controls (Senior et al. 1999). 6. In the year 2003, our group has shown that anti-ESRRAL Proteus antibodies from patients with active RA can haemolyse sheep red blood cells (SRBC) coated with HLA-DR4/1-containing "EQRRAA" but not HLA-B27-containing "QTDRED" amino acid synthetic peptides more frequently than patients with AS and healthy controls. By contrast, active AS patients with high anti-QTDRED Klebsiella antibodies had higher levels of an in vitro haemolytic reactions on SRBCs coated with "QTDRED" but not "ESRRAL" synthetic peptides when compared to patients with RA or to healthy controls . Hence, RA and AS groups of patients could behave as reciprocal controls to each other concerning the involvement of antibodies against Proteus antigens in RA and Klebsiella antigens in AS. Blood donors did not react against either Proteus or Klebsiella antigens. 7. Between the year 1985 and 2003, many serological studies have been carried out by various independent groups. Cumulatively, they show that elevated antibodies against various antigens from Proteus mirabilis were observed in more than 1350 RA patients from 15 different countries worldwide when compared to corresponding healthy controls . 8. In the year 2004, a collaborative study was carried out involving Finish and Japanese patients with RA. It was observed that these patients were all showing significantly elevated levels of IgG antibodies to whole and synthetic peptide antigen preparations from P. mirabilis but not to those from E. coli and S. marcescens bacteria when compared to corresponding healthy controls or patients with systemic lupus erythematosus (Rashid et al. 2004). 9. In a recent multi-centre prospective study, it has been shown that rheumatoid factor-(RF) positive patients with RA had significantly elevated levels of IgM and IgA antibodies against Proteus mirabilis and IgA antibodies against E. coli when compared to disease and healthy control subjects  Tiwana et al. (1999) In vitro and in vivo immunological cross-reactivities between antibodies against P. mirabilis and self-antigenic peptide molecules.
ELISA and peptide binding dilution assay Rashid et al. (1999) Elevated antibodies to P. mirabilis in Spanish and Norwegian patients with RA when compared to corresponding HCs.
IIF (Newkirk et al. 2005). However, many other studies carried out previously had shown that only anti-Proteus but no anti-E. coli antibodies were observed to be elevated in RA patients , Subair et al. 1995Tiwana et al. 1996;Tani et al. 1997;Rashid et al. 2004). 10. In another study carried out by a group from Los Angeles, RA patients were reported to have IgA anti-Proteus antibodies detectable more than in healthy controls. However, several molecules from this microbe had shown reduced IgA immune responses compared to controls (Weisbart et al. 2005). It has been suggested that the occurrence of some "holes" in the IgA immune repertoire for certain antigens in Proteus bacteria could explain the possibility that RA patients are more liable to harbour this microbe and to have more frequent bouts of (albeit, sub-clinical) infections, with a consequent intermittent and/or progressive enhancement of anti-Proteus antibody responses. 11. Most recently, our group has studied the humoral immune responses against a new antigenic moiety from Proteus urease F enzyme, which was found to be non homologous to RA-targeted synovial tissue structures. We have shown that active RA patients had significantly elevated IgG and IgM anti-Proteus peptide antibodies when compared to healthy controls (Rashid et al. submitted). This latter finding argues against the suggestion that elevated antibodies against the Proteus crossreactive antigens in RA patients could be an epiphenomenona resulting from increased exposure of auto-antigens due to pathological damages produced by bacterial triggers.

Proteus urinary tract infections and RA
Various microbiological and immunological data results support the suggestion that there is a link between RA and urinary tract infections (UTI) mainly caused by Proteus (Table II). Patients with RA were reported to have a higher frequency of recurrent UTIs (Lawson and Maclean 1966;Tishler et al. 1992). Urine samples from RA patients yield higher isolation rates of P. mirabilis microbes than patients with osteoarthritis or healthy controls (Ebringer et al. 1993). RA patients had higher levels of antibodies against Proteus in their urine when compared to those of healthy subjects (Senior et al. 1999) and significant correlation was detected between serum anti-Proteus antibodies and Proteus urinary isolation rates in RA patients when compared to healthy controls . Furthermore, RA patients group who were receiving a vegetarian diet had a drop in the levels of antibodies against P. mirabilis but not E. coli, when compared to those patients who were omnivores (Kjeldsen-Kragh et al. 1995). This dietary treatment may have exerted its specific anti-microbial effect via the actions of lignans and phytoestrogen metabolites (Adlercreutz et al.  Senior et al. (1999) Sub clinical Proteus bacteriuria and elevated levels of anti-Proteus antibodies in urine and serum of patients with RA when compared to HCs.
ELISA and Immunoblot Ushakova et al. (2000) Elevated IgM and IgG antibodies to P. mirabilis in Russian patients with RA when compared to HCs.
ELISA Wilson et al. (2003) In vitro immunological cytotoxicity reaction occurring between anti-Proteus and anti-HLA-DRB1 antibodies with the corresponding crossreactive peptide molecules.
ELISA and sheep red cell haemolytic assays Rashid et al. (2004) Finish and Japanese patients with RA had elevated antibodies against whole and synthetic peptide antigenic preparations from P. mirabilis but not those from E. coli and S. marcescens when compared to corresponding HCs.
ELISA and IIF Newkirk et al. (2005) Elevated IgM and IgA antibodies to P. mirabilis and IgA antibodies to E. coli but not to 6 other bacterial and viral agents in rheumatoid factor positive RA patients when compared to those with other rheumatic diseases.
1986), which are known to possess anti-bacterial activity in vitro (Ito et al. 1982).
Females are more vulnerable to develop UTIs than males and up to 60% of women have been reported to develop at least one episode of UTI in their lifetime (Foxman et al. 2000). A significant number of these women might have recurrent infections, and the chance of infection by Proteus species was found to increase with age (Nicolle 1996).
The increased incidence of UTIs in RA patients and the more frequent occurrence of UTIs in females, especially those of middle age and elderly groups might give an answer to the higher prevalence of RA among women.

Molecular mimicry as a plausible aetiopathogenetic mechanism
Molecular and immunological inter-relation between the triggering aetiological factor, namely Proteus mirabilis antigens, and targeted synovial tissue structures expressing HLA molecules that contain "SE" amino acid motif and collagen type XI, in RA could be explained by the molecular mimicry or cross-reactivity mechanism (Ebringer et al. 2005). High antibody titres against Proteus haemolysin and urease antigens in patients with active RA could bind to cross-reactive self antigens and consequently result in production of various inflammatory and immunological damaging mediators based on the mechanism of antibody-mediated cytotoxicity reactions (Figure 1). Damage to the synovial and other joint structures could result in the release of self-antigenic particles and auto-antibody production. Anti-Proteus crossreactive antibodies, which might result from recurrent, albeit subclinical UTIs, together with high levels of secondary auto-antibodies will bind to RA-targeted antigens in the synovial tissues. These immunological reactions could coincide with the initiation of clinical and laboratory exacerbations and further pathological damages.

Proposal of a new therapeutic strategy
Currently patients with RA are treated with various therapeutic strategies involving the use of antiinflammatory, immunosuppressive and biological agents as well as non-medical treatment modalities (Genovese and Harris 2005).
Although, the current medical treatments, especially those involving anti-tumour necrosis factor therapies (Haraoui 2005), have a promising beneficial effect particularly in easing or even halting the disease progress, they are expensive (Merkesdal et al. 2004) and cause side-effects. In order, to achieve a prolonged clinical remission, continuous immunosuppression with these drugs is required because this kind of treatment could not eradicate the causative microbial agents.
Based on the existing evidence for involving Proteus bacteria in the pathogenesis of RA, it is logical to propose a new therapeutic modality in the management of RA, which could be implemented in conjunction with other currently used treatments. This new treatment includes anti-Proteus measures involving the use of Proteus-sensitive antibiotics with dietary manipulations in the forms of vegetarian diet (Kjeldsen-Kragh et al. 1995) and high daily intake of water and fruit juices containing fructose such as cranberry juice (Rashid et al. 2001).
It could also be possible to make a vaccine mainly derived from Proteus antigenic molecules that do not contain the cross-reactive epitopes, in order to prevent susceptible individuals of acquiring Proteus UTIs and decrease the chance of developing RA or at least limit further damages in those with established disease.

Conclusion
Based on the results of various studies carried out in relation to Proteus microbes, it could be said that compelling evidence exists linking this microbe to RA, starting with recurrent sub-clinical Proteus UTIs and ending in the full development of RA. To prove the scientific logic of this possibility, and its benefit to patients clinical trials using anti-Proteus measures in RA are required to be carried out in prospective longitudinal studies. 1) Proteus mirabilis bacteria were isolated more frequently in patients with RA than those with other disease and healthy controls (Wilson et al. 1990;Senior et al. 1999). 2) A positive correlation has been detected between anti-Proteus antibodies and the rate of isolation of these microbes from urine of RA patients when compared to healthy subjects .
3) RA patients receiving vegetarian diet are more liable to have decreased levels of anti-Proteus antibodies than omnivores (Kjeldsen-Kragh et al. 1995). 4) Elevated levels of anti-Proteus antibodies were observed in sera of RA patients coming from 15 different countries .

Indirect evidence:
1) Patients with severe RA had a higher frequency of recurrent UTIs (Tishler et al. 1992).
2) Women are more liable to develop recurrent UTIs (Franco 2005). This finding in combination to point number 1 above, could explain why RA is more common among women than men.
3) Proteus bacteria, which are urease-positive, account for 15% of UTIs, and affect the upper urinary tract, especially kidneys, whilst E. coli, another urogenic but urease-negative bacteria, account for the majority of UTIs and mainly involve bladder (Cattell 2005). 4) Patients with recurrent UTIs are more likely to respond to high daily intake of cranberry juice (Raz et al. 2004). Whether cranberry juice could have a beneficial effect in patients with RA needs to be investigated in a prospective controlled study.