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Bacteriophages in autoimmune disease and other inflammatory conditions
Medical Hypotheses (2004) 62, 493–498
http://intl.elsevierhealth.com/journals/mehy
Bacteriophages in autoimmune disease and other inflammatory conditions Peter A. Riley* Department of Medical Microbiology, St. George’s Hospital, Blackshaw Road, London SW17 0QT, UK Received 11 June 2003; accepted 9 December 2003
Summary There are several autoimmune diseases and other inflammatory conditions where an infectious aetiology is suggested by the epidemiology, clinical course and pathological findings. Many candidate bacteria and viruses have been considered as potential aetiological agents but mostly without firm proof. Bacteriophages are viruses that infect bacteria and may be found wherever bacteria are located, but would not be detected unless specifically sought. They have not previously been considered to be pathogens. Bacteriophages are immunogenic and therefore could play a role in the pathogenesis of autoimmune and other inflammatory diseases by acting as antigens on epithelial surfaces, bound to antibody as immune complexes, through molecular mimicry or possibly as superantigens. c 2004 Elsevier Ltd. All rights reserved.
The role of microbes in the aetiology of autoimmune disease and other inflammatory conditions There are several autoimmune diseases and other inflammatory conditions where an infectious aetiology is suggested by the epidemiology, clinical course and pathological findings. It is now generally accepted that commensal bacteria play a role in the pathogenesis of inflammatory bowel disease [1–3]. Experimental data have shown that HLA-B27 transgenic rats spontaneously develop colitis and arthritis, however when they are bred in a germfree environment, no or attenuated inflammation occurs [4]. Animals that are initially germ-free and disease-free develop inflammation if they are subsequently colonised with bacteria [5]. The exact mechanism whereby inflammation occurs has *
Tel.: +44-020-8725-5707; fax: +44-020-8725-5694. E-mail address: [email protected] (P.A. Riley).
not been determined but it is believed that an abnormal immune response to normal flora is involved and there is some evidence to support a breakdown in tolerance [6]. In humans, Escherichia coli has been implicated, with certain strains being more likely to be found in patients with disease compared to healthy controls [7,8]. Attempts have been made to treat ulcerative colitis by replacing the resident E. coli with benign strains, with some success [9]. Patients with ulcerative colitis induced pouchitis have also been treated using a combination of lactobacilli, bifidobacteria and streptococci [10]. Many infectious organisms have been postulated as the aetiological agent of rheumatoid arthritis [11], and Klebsiella has been proposed to play a role in the pathogenesis of ankylosing spondylitis [12]. There is evidence that sarcoidosis has an infectious aetiology [13], and Propionibacterium acnes has been proposed as a candidate agent [14]. Numerous viruses and bacteria have also been proposed as aetiological agents in multiple sclerosis
0306-9877/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2003.12.016
494 [15,16]. Atherosclerosis has long been known to have an inflammatory component to its pathogenesis and again bacteria have been implicated, but without certain proof [17]. Bacterial superantigens may be involved in autoantibody production in connective tissue diseases such as SLE [18]. There are many features of Kawasaki disease that suggest an infectious aetiology [19], and various organisms including P. acnes have been investigated [20]. Bacterial superantigens have also been suggested as possible factors responsible for Kawasaki disease [21]. All these conditions are similar in that there is some evidence of a bacterial aetiology but there is no certain proof. Is it possible that these conditions are triggered by something that is associated with the bacteria but has previously been overlooked as an antigen? Wherever bacteria are found, bacteriophages may also be present, but they will not be detected unless specifically sought, since they do not grow on conventional media or infect conventional cell cultures used for virus isolation. The evidence that some autoimmune diseases do not develop in a germ-free environment is intriguing since germ-free environments are also bacteriophage-free. Could bacteriophages associated with the incriminated bacteria be the actual aetiological agents? Humans are extensively colonised with bacteria on the skin, upper respiratory tract, lower intestinal tract and the lower female genital tract. In fact there are more bacteria in and on us, than the total number of cells that make up our own bodies [22]. These bacteria are acquired soon after birth usually from the mother and also the immediate environment. The composition of the normal flora is not stable and may be influenced by many factors e.g. exposure to antibiotics. New organisms may be acquired from contact with other humans and animals and from food and it has been shown that successive waves of colonisation with E. coli of different biotypes occur [23]. New bacteria may bring bacteriophages with them. Bacteriophages are known to be immunogenic and have even been used to assess humoral immune function [24] but more extensive studies of immune response to bacteriophages have not been conducted.
Structure and genetics of bacteriophages Bacteriophages, often referred to simply as phages, are viruses that infect bacteria. They are relatively simple structures and consist of nucleic
Riley acid contained within a protein coat. As in conventional viruses, the nucleic acid may be either DNA or RNA but not both. The protein coat or capsid is constructed from a large number of subunits or capsomeres. These capsomeres may be made up of single subunits or groups of subunits and are organised either helically to form a hollow tube as seen in the filamentous bacteriophages or they may form near-spherical hollows. Perhaps the best studied phages are those that infect E. coli and other coliform bacteria and these are sometimes referred to as coliphages. Examples include the filamentous phages f1, fd and M13 or the more complex T4 phage. Bacteriophages are probably associated with all bacterial families, however a specific phage usually has a limited host range. Some are able to attack related bacteria of different species e.g. E. coli and Shigella sonnei whereas others may only be able to infect certain strains of individual bacterial species. When they infect bacteria, phages bind to specific receptors on the bacterial surface and this is one reason why there is host restriction as different phages will bind to different receptors. Following attachment, phage nucleic acid enters the cell. What happens next depends upon several factors. During the lytic cycle, phage genes are expressed almost immediately. The result is replication of phage nucleic acid and production of capsid protein. The phage particles are then assembled, the cell lyses and large numbers of infectious phage particles are released. Phages that go through this lytic cycle are said to be virulent and lysis results in cell death. On the other hand, temperate phages do not always produce a lytic cycle and can form a stable relationship, termed lysogeny, with the host. The phage genes are inserted into the host chromosome at a specific site and is known as the prophage. There is no replication of phage nucleic acid or production of phage capsid protein. A lysogenic bacterium may be induced to enter the lytic cycle and thus liberate infectious phage. Inducing agents include ultra-violet light and a variety of carcinogens and mutagens that act on repressor proteins [25]. Bacteriophages are known to be immunogenic but during lysogeny a phage will not be seen by the immune system as there is no production of phage protein because of gene suppression. Bacteriophages do not infect or replicate within human cells and have not been considered to be pathogens. However, there are instances when bacteriophages play a part in the pathogenesis of bacterial infections e.g. phage conversion seen Streptococcus pyogenes strains that cause scarlet
Bacteriophages in autoimmune disease and other inflammatory conditions fever and toxigenic Corynebacterium diphtheriae. The toxin genes are not located on the bacterial chromosome but carried by a temperate bacteriophage. Recently, it has been hypothesised that bacteriophages of lactobacilli may be responsible for the pathogenesis of bacterial vaginosis [26]. The vagina is normally colonised with lactobacilli which are believed to have a protective role suppressing the growth of anaerobic bacteria. Phage mediated lysis of lactobacilli may result in a reduction of the normal lactobacillus flora permitting subsequent overgrowth of anaerobes. Lysogenic phages have been found in lactobacilli isolated from the vagina and factors affecting phage induction in lysogenic strains have also been studied. Lysis is strongly stimulated by the cigarette carcinogen benzol(a)pyrone diol expoxide [27]. Bacterial vaginosis is commoner in smokers and in those who have partners who smoke [26].
Hypothesis My hypothesis is that bacteriophages may play a role as antigens in the pathogenesis of autoimmune disease and inflammatory conditions. Candidate diseases that could be explained by the hypothesis are inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis and other connective tissue disorders such as SLE, as well as
Figure 1
495
multiple sclerosis, atherosclerosis and Kawasaki disease. Lytic phages may be acquired when new bacteria carrying phages are introduced into the normal flora. Lytic phage infection of the normal flora would result in release of large amounts of phage protein (Fig. 1). Alternatively, a lytic cycle may be induced in lysogenic strains already resident in the normal flora following possible exposure to inducing agents in the environment e.g. cigarette smoke or in foods (Fig. 2). The phage capsid proteins are antigenic and large amounts of antigenic protein released could result in an immune response and release of pro-inflammatory cytokines with various possible consequences (Fig. 3). Large amounts of antigen released onto epithelial surfaces of the gut for example may lead to inflammatory bowel disease or if released at normally sterile sites e.g. joints, would cause inflammation at these sites. Immune complexes of phage antigen and antibody might be deposited locally or at distant sites and cause damage as part of a type III hypersensitivity reaction. Another possibility is that phage protein may share determinants with a host proteins i.e., molecular mimicry. Antibody or cell mediated immune response to the phage antigen would also be directed at the host antigen with resultant tissue damage. This mechanism might explain the production of autoantibodies in the wide range of diseases previously discussed. Could phage antigens also act as superantigens? Superantigens
Acquisition of lytic bacteriophage and effect on normal bacterial flora.
496
Riley
Figure 2
Induction in lysogenic bacteriophages in normal bacterial flora after exposure to inducing agent.
Figure 3
Possible immunological consequences after release of bacteriophage antigen.
are able to stimulate vast numbers of T cells by directly binding class II molecules of antigenpresenting cells to T-cell receptors and may explain some diseases such as Kawasaki disease or play a part in autoantibody production, but as yet there is no evidence to suggest that phages may do this. Relapses of disease, for example that seen in inflammatory bowel disease, may occur when new waves of lysis occur following reintroduction of
virulent phages to the bacterial flora or re-exposure to agents that induce lysis in lysogenic organisms.
Published data supporting the hypothesis A recent study has demonstrated that patients with abdominal aortic aneurysm are more likely to have
Bacteriophages in autoimmune disease and other inflammatory conditions been infected with strains of Chlamydia pneumoniae that harbour phages rather than strains without phages [28]. The phage-containing strains were identified by the detection in the patients’ sera of antibodies against phage capsid. The authors speculated whether the phage containing strains of C. pneumoniae are more virulent presumably by phage conversion, but no genes for a putative virulence factor have been identified in the phage genome. Perhaps the phage capsid itself, which is immunogenic, is the pathogenic factor? Further evidence is that phage-containing strains of C. pneumioniae [29–31], but not phage-free strains [32,33], can exacerbate the development of atherosclerosis in LDR = or ApoE = mice. In the case of inflammatory bowel disease the phage theory could be used to explain the benefit of probiotic therapy [9,10]; normal flora with lysogenic phages or flora susceptible to a lytic phage may be replaced with strains that are resistant to phage infection or induction of lysis. Another interesting observation is that smoking is commoner in those with Crohn’s disease [2,3]. Could inducing agents in cigarette smoke precipitate lytic cycles, thus leading to inflammation? This would not however, explain why smoking appears to be protective in ulcerative colitis. The exact mechanisms of the effects of smoking in inflammatory bowel disease have yet to be fully elucidated [34], but recently published work has demonstrated that the nicotinic acetylcholine receptor alpha7 subunit is required for acetylcholine inhibition of macrophage tumour-necrosis factor release, a potent inflammatory mediator [35]. This would explain the protective effects of smoking in ulcerative colitis but not why Crohn’s disease is commoner in smokers.
Testing the hypothesis The hypothesis is testable but there may be difficulties. Panels of phage antigen could be screened against sera from patients with candidate inflammatory or autoimmune conditions, or screened for induction of T-cell proliferation or for their ability to act as superantigens. The difficulty would be choosing which of the potentially hundreds of phages might be likely candidates. If molecular mimicry is the mechanism, the number of likely phages could be narrowed down by comparison of host and phage gene sequences, if known. Candidate phages could then be tested in an animal model, but phages that may occur in bacteria found in humans may not act in the same way in a different animal species.
497
Conclusion The hypothesis accounts for why bacteria have been implicated in the aetiology of some autoimmune and inflammatory conditions and why no proof of a pathogenic role for these bacteria has been proven. The hypothesis describes how bacteria may act as vectors for the actual aetiological agents, bacteriophages. The bacteriophages may cause disease as new antigens arriving with new bacteria, or be revealed as new antigens following induction in lysogenic organisms within the normal bacterial flora.
References [1] Campieri M, Gionchetti P. Bacteria as the cause of ulcerative colitis. Gut 2001;48:132–5. [2] Hendrickson BA, Gokhale R, Cho JH. Clinical aspects and pathophysiology of inflammatory bowel disease. Clin Microbiol Rev 2002;15:79–94. [3] Podolsky DK. Inflammatory bowel disease. N Eng J Med 2002;347:417–29. [4] Taurog JD, Richmanson JA, Croft JT, Simmons WA, Zhou M, Fernandez-Sueiro JL, et al. The germfree state prevents development of gut and joint inflammatory disease in HLAB27 transgenic rats. J Exp Med 1994;180:2359–64. [5] Rath HC, Schultz M, Freitag R, et al. Different subsets of enteric bacteria induce and perpetuate experimental colitis in rats and mice. Infect Immun 2001;69:2277–85. [6] Duchmann R, Kaiser I, Hermann E, Mayet W, Ewe K, Meyer zum Buschenfelde KH. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD). Clin Exp Immunol 1995;102:448–55. [7] Cooke EM. Properties of strains of Escherichia coli isolated from the faeces of patients with ulcerative colitis, patients with diarrhoea and normal persons. J Pathol Bacteriol 1968;95:101–13. [8] Burke FA, Axon ATR. Ulcerative colitis and Escherichia coli with adhesive properties. J Clin Pathol 1988;40:782–6. [9] Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DM, Axon ATR. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 1999;354:635–9. [10] Gionchetti P, Rizzello F, Venturi A, et al. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology 2000;119:305–9. [11] Kingsley G. Microbial DNA in the synovium – a role in aetiology or a mere bystander. Lancet 1997;349:782–6. [12] Toivanen P, Hansen DS, Mestre F, et al. Somatic serogroups, capsular types, and species of faecal Klebsiella in patients with ankylosing spondylitis. J Clin Microbiol 1999;37: 2808–12. [13] James DG. Etiology of sarcoidosis. Sarcoidosis 1994; 11(Suppl 1):43–51. [14] Ishige I, Usui Y, Takemura T, Eishi Y. Quantitative PCR of mycobacterial and propionibacterial DNA in lymph nodes of japanese patients with sarcoidosis. Lancet 1999;354: 120–3. [15] Wolfson C, Talbot P. Bacterial infection as a cause of multiple sclerosis. Lancet 2002;360:352.
498 [16] Murray J. Infection as a cause of multiple sclerosis. BMJ 2002;325:1128. [17] Boman J, Hammerschlag MR. Chlamydia pneumoniae and atherosclerosis: critical assessment of diagnostic methods and relevance to treatment studies. Clin Microbiol Rev 2002;15:1–20. [18] Friedman SM, Posnett DN, Tumang JR, Cole BC, Crow MK. A potential role for microbial superantigens in the pathogenesis of systemic autoimmune disease. Arthritis Rheum 1991;34:468–80. [19] Yanagawa H, Nakamura Y, Yashiro M, et al. A nationwide incidence survey of Kawasaki disease in 1985–1986 in Japan. J Infect Dis 1988;158:1296–301. [20] Kato H, Fujimoto T, Inoue O, et al. Variant strain of Propionibacterium acnes: a clue to the aetiology of Kawasaki disease. Lancet 1983;ii:1383–8. [21] Levin M, Tizard EJ, Dillon MJ. Kawasaki disease; recent advances. Arch Dis Child 1991;66:1369–72. [22] Mims C, Nash A, Stephen J. In: Mims’ pathogenesis of infectious disease. 5th ed. London: Academic Press; 2001. p. 47. [23] Hinton M, Hampson DJ, Hampson E, Linton AH. A comparison of the ecology of Escherichia coli in the intestine of healthy unweaned pigs and pigs after weaning. J Appl Bacteriol 1985;58:471–7. [24] Pyun KH, Ochs HD, Wedgwood RJ, Yang XQ, Heller SR, Reimer CB. Human antibody responses to bacteriophage phi X 174: sequential induction of IgM and IgG subclass antibody. Clin Immunol Immunopathol 1989;51:252–63. [25] Bennet PM, Howe TGB. Bacterial and bacteriophage genetics. In: Balows A, Duerden BI, editors. Topley and Wilson’s microbiology and microbial infections, vol 2. Systematic bacteriology. 9th ed. London: Arnold. 1998, p. 231–294. [26] Blackwell AL. Vaginal bacterial phaginosis? Sex Transm Inf 1999;75:352–3.
Riley [27] Tao L, Pavlova SI, Mou SM. Factors affecting phage induction in vaginal lactobacilli. In: Abstracts from the annual meeting of the international infectious disease society for obstetrics and gynecology USA, Las Vegas, Nevada, 25–26 April, 1997. Infect Dis Obstet Gynecol 1997;5:66. [28] Karunakaran KP, Blanchard JF, Raudonikiene A, Shen C, Murdin AD, Brunham RC. Molecular detection and seroepidemiology of the Chlamydia pneumoniae bacteriophage (UCpn1). J Clin Microbiol 2002;40:4010–4. [29] Hu H, Pierce GN, Zhong G. The atherogenic effects of chlamydia are dependent on serum cholesterol and specific to Chlamydia pneumoniae. J Clin Invest 1999;103:747– 53. [30] Lu L, Hu H, Ji H, Murdin AD, Pierce GN, Zhong G. Chlamydia pneumoniae infection significantly exacerbates aortic atherosclerosis in an LDLR-=- mouse model within six months. Mol Cell Biochem 2000;215:123–8. [31] Moazed TC, Campbell LA, Rosenfeld ME, Grayston JT, Kuo CC. Chlamydia pneumoniae infection accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice. J Infect Dis 1999;180:238–41. [32] Aalto-Setala K, Laitinen K, Erkkila L, et al. Chlamydia pneumoniae does not increase atherosclerosis in the aortic root of apolipoprotein E-deficient mice. Arterioscl Throm Vas 2001;21:578–84. [33] Caligiuri G, Rottenberg M, Nicoletti A, Wigzell H, Hansson GK. Chlamydia pneumoniae infection in mice does not induce or modify atherosclerosis in mice. Circulation 2001;103:2834–8. [34] Rubin DT, Hanauer SB. Smoking and inflammatory bowel disease. Eur J Gastroen Hepat 2000;12:855–62. [35] Wang H, Yu M, Ochani M, et al. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 2003;421:384–8.
http://intl.elsevierhealth.com/journals/mehy
Bacteriophages in autoimmune disease and other inflammatory conditions Peter A. Riley* Department of Medical Microbiology, St. George’s Hospital, Blackshaw Road, London SW17 0QT, UK Received 11 June 2003; accepted 9 December 2003
Summary There are several autoimmune diseases and other inflammatory conditions where an infectious aetiology is suggested by the epidemiology, clinical course and pathological findings. Many candidate bacteria and viruses have been considered as potential aetiological agents but mostly without firm proof. Bacteriophages are viruses that infect bacteria and may be found wherever bacteria are located, but would not be detected unless specifically sought. They have not previously been considered to be pathogens. Bacteriophages are immunogenic and therefore could play a role in the pathogenesis of autoimmune and other inflammatory diseases by acting as antigens on epithelial surfaces, bound to antibody as immune complexes, through molecular mimicry or possibly as superantigens. c 2004 Elsevier Ltd. All rights reserved.
The role of microbes in the aetiology of autoimmune disease and other inflammatory conditions There are several autoimmune diseases and other inflammatory conditions where an infectious aetiology is suggested by the epidemiology, clinical course and pathological findings. It is now generally accepted that commensal bacteria play a role in the pathogenesis of inflammatory bowel disease [1–3]. Experimental data have shown that HLA-B27 transgenic rats spontaneously develop colitis and arthritis, however when they are bred in a germfree environment, no or attenuated inflammation occurs [4]. Animals that are initially germ-free and disease-free develop inflammation if they are subsequently colonised with bacteria [5]. The exact mechanism whereby inflammation occurs has *
Tel.: +44-020-8725-5707; fax: +44-020-8725-5694. E-mail address: [email protected] (P.A. Riley).
not been determined but it is believed that an abnormal immune response to normal flora is involved and there is some evidence to support a breakdown in tolerance [6]. In humans, Escherichia coli has been implicated, with certain strains being more likely to be found in patients with disease compared to healthy controls [7,8]. Attempts have been made to treat ulcerative colitis by replacing the resident E. coli with benign strains, with some success [9]. Patients with ulcerative colitis induced pouchitis have also been treated using a combination of lactobacilli, bifidobacteria and streptococci [10]. Many infectious organisms have been postulated as the aetiological agent of rheumatoid arthritis [11], and Klebsiella has been proposed to play a role in the pathogenesis of ankylosing spondylitis [12]. There is evidence that sarcoidosis has an infectious aetiology [13], and Propionibacterium acnes has been proposed as a candidate agent [14]. Numerous viruses and bacteria have also been proposed as aetiological agents in multiple sclerosis
0306-9877/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2003.12.016
494 [15,16]. Atherosclerosis has long been known to have an inflammatory component to its pathogenesis and again bacteria have been implicated, but without certain proof [17]. Bacterial superantigens may be involved in autoantibody production in connective tissue diseases such as SLE [18]. There are many features of Kawasaki disease that suggest an infectious aetiology [19], and various organisms including P. acnes have been investigated [20]. Bacterial superantigens have also been suggested as possible factors responsible for Kawasaki disease [21]. All these conditions are similar in that there is some evidence of a bacterial aetiology but there is no certain proof. Is it possible that these conditions are triggered by something that is associated with the bacteria but has previously been overlooked as an antigen? Wherever bacteria are found, bacteriophages may also be present, but they will not be detected unless specifically sought, since they do not grow on conventional media or infect conventional cell cultures used for virus isolation. The evidence that some autoimmune diseases do not develop in a germ-free environment is intriguing since germ-free environments are also bacteriophage-free. Could bacteriophages associated with the incriminated bacteria be the actual aetiological agents? Humans are extensively colonised with bacteria on the skin, upper respiratory tract, lower intestinal tract and the lower female genital tract. In fact there are more bacteria in and on us, than the total number of cells that make up our own bodies [22]. These bacteria are acquired soon after birth usually from the mother and also the immediate environment. The composition of the normal flora is not stable and may be influenced by many factors e.g. exposure to antibiotics. New organisms may be acquired from contact with other humans and animals and from food and it has been shown that successive waves of colonisation with E. coli of different biotypes occur [23]. New bacteria may bring bacteriophages with them. Bacteriophages are known to be immunogenic and have even been used to assess humoral immune function [24] but more extensive studies of immune response to bacteriophages have not been conducted.
Structure and genetics of bacteriophages Bacteriophages, often referred to simply as phages, are viruses that infect bacteria. They are relatively simple structures and consist of nucleic
Riley acid contained within a protein coat. As in conventional viruses, the nucleic acid may be either DNA or RNA but not both. The protein coat or capsid is constructed from a large number of subunits or capsomeres. These capsomeres may be made up of single subunits or groups of subunits and are organised either helically to form a hollow tube as seen in the filamentous bacteriophages or they may form near-spherical hollows. Perhaps the best studied phages are those that infect E. coli and other coliform bacteria and these are sometimes referred to as coliphages. Examples include the filamentous phages f1, fd and M13 or the more complex T4 phage. Bacteriophages are probably associated with all bacterial families, however a specific phage usually has a limited host range. Some are able to attack related bacteria of different species e.g. E. coli and Shigella sonnei whereas others may only be able to infect certain strains of individual bacterial species. When they infect bacteria, phages bind to specific receptors on the bacterial surface and this is one reason why there is host restriction as different phages will bind to different receptors. Following attachment, phage nucleic acid enters the cell. What happens next depends upon several factors. During the lytic cycle, phage genes are expressed almost immediately. The result is replication of phage nucleic acid and production of capsid protein. The phage particles are then assembled, the cell lyses and large numbers of infectious phage particles are released. Phages that go through this lytic cycle are said to be virulent and lysis results in cell death. On the other hand, temperate phages do not always produce a lytic cycle and can form a stable relationship, termed lysogeny, with the host. The phage genes are inserted into the host chromosome at a specific site and is known as the prophage. There is no replication of phage nucleic acid or production of phage capsid protein. A lysogenic bacterium may be induced to enter the lytic cycle and thus liberate infectious phage. Inducing agents include ultra-violet light and a variety of carcinogens and mutagens that act on repressor proteins [25]. Bacteriophages are known to be immunogenic but during lysogeny a phage will not be seen by the immune system as there is no production of phage protein because of gene suppression. Bacteriophages do not infect or replicate within human cells and have not been considered to be pathogens. However, there are instances when bacteriophages play a part in the pathogenesis of bacterial infections e.g. phage conversion seen Streptococcus pyogenes strains that cause scarlet
Bacteriophages in autoimmune disease and other inflammatory conditions fever and toxigenic Corynebacterium diphtheriae. The toxin genes are not located on the bacterial chromosome but carried by a temperate bacteriophage. Recently, it has been hypothesised that bacteriophages of lactobacilli may be responsible for the pathogenesis of bacterial vaginosis [26]. The vagina is normally colonised with lactobacilli which are believed to have a protective role suppressing the growth of anaerobic bacteria. Phage mediated lysis of lactobacilli may result in a reduction of the normal lactobacillus flora permitting subsequent overgrowth of anaerobes. Lysogenic phages have been found in lactobacilli isolated from the vagina and factors affecting phage induction in lysogenic strains have also been studied. Lysis is strongly stimulated by the cigarette carcinogen benzol(a)pyrone diol expoxide [27]. Bacterial vaginosis is commoner in smokers and in those who have partners who smoke [26].
Hypothesis My hypothesis is that bacteriophages may play a role as antigens in the pathogenesis of autoimmune disease and inflammatory conditions. Candidate diseases that could be explained by the hypothesis are inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis and other connective tissue disorders such as SLE, as well as
Figure 1
495
multiple sclerosis, atherosclerosis and Kawasaki disease. Lytic phages may be acquired when new bacteria carrying phages are introduced into the normal flora. Lytic phage infection of the normal flora would result in release of large amounts of phage protein (Fig. 1). Alternatively, a lytic cycle may be induced in lysogenic strains already resident in the normal flora following possible exposure to inducing agents in the environment e.g. cigarette smoke or in foods (Fig. 2). The phage capsid proteins are antigenic and large amounts of antigenic protein released could result in an immune response and release of pro-inflammatory cytokines with various possible consequences (Fig. 3). Large amounts of antigen released onto epithelial surfaces of the gut for example may lead to inflammatory bowel disease or if released at normally sterile sites e.g. joints, would cause inflammation at these sites. Immune complexes of phage antigen and antibody might be deposited locally or at distant sites and cause damage as part of a type III hypersensitivity reaction. Another possibility is that phage protein may share determinants with a host proteins i.e., molecular mimicry. Antibody or cell mediated immune response to the phage antigen would also be directed at the host antigen with resultant tissue damage. This mechanism might explain the production of autoantibodies in the wide range of diseases previously discussed. Could phage antigens also act as superantigens? Superantigens
Acquisition of lytic bacteriophage and effect on normal bacterial flora.
496
Riley
Figure 2
Induction in lysogenic bacteriophages in normal bacterial flora after exposure to inducing agent.
Figure 3
Possible immunological consequences after release of bacteriophage antigen.
are able to stimulate vast numbers of T cells by directly binding class II molecules of antigenpresenting cells to T-cell receptors and may explain some diseases such as Kawasaki disease or play a part in autoantibody production, but as yet there is no evidence to suggest that phages may do this. Relapses of disease, for example that seen in inflammatory bowel disease, may occur when new waves of lysis occur following reintroduction of
virulent phages to the bacterial flora or re-exposure to agents that induce lysis in lysogenic organisms.
Published data supporting the hypothesis A recent study has demonstrated that patients with abdominal aortic aneurysm are more likely to have
Bacteriophages in autoimmune disease and other inflammatory conditions been infected with strains of Chlamydia pneumoniae that harbour phages rather than strains without phages [28]. The phage-containing strains were identified by the detection in the patients’ sera of antibodies against phage capsid. The authors speculated whether the phage containing strains of C. pneumoniae are more virulent presumably by phage conversion, but no genes for a putative virulence factor have been identified in the phage genome. Perhaps the phage capsid itself, which is immunogenic, is the pathogenic factor? Further evidence is that phage-containing strains of C. pneumioniae [29–31], but not phage-free strains [32,33], can exacerbate the development of atherosclerosis in LDR = or ApoE = mice. In the case of inflammatory bowel disease the phage theory could be used to explain the benefit of probiotic therapy [9,10]; normal flora with lysogenic phages or flora susceptible to a lytic phage may be replaced with strains that are resistant to phage infection or induction of lysis. Another interesting observation is that smoking is commoner in those with Crohn’s disease [2,3]. Could inducing agents in cigarette smoke precipitate lytic cycles, thus leading to inflammation? This would not however, explain why smoking appears to be protective in ulcerative colitis. The exact mechanisms of the effects of smoking in inflammatory bowel disease have yet to be fully elucidated [34], but recently published work has demonstrated that the nicotinic acetylcholine receptor alpha7 subunit is required for acetylcholine inhibition of macrophage tumour-necrosis factor release, a potent inflammatory mediator [35]. This would explain the protective effects of smoking in ulcerative colitis but not why Crohn’s disease is commoner in smokers.
Testing the hypothesis The hypothesis is testable but there may be difficulties. Panels of phage antigen could be screened against sera from patients with candidate inflammatory or autoimmune conditions, or screened for induction of T-cell proliferation or for their ability to act as superantigens. The difficulty would be choosing which of the potentially hundreds of phages might be likely candidates. If molecular mimicry is the mechanism, the number of likely phages could be narrowed down by comparison of host and phage gene sequences, if known. Candidate phages could then be tested in an animal model, but phages that may occur in bacteria found in humans may not act in the same way in a different animal species.
497
Conclusion The hypothesis accounts for why bacteria have been implicated in the aetiology of some autoimmune and inflammatory conditions and why no proof of a pathogenic role for these bacteria has been proven. The hypothesis describes how bacteria may act as vectors for the actual aetiological agents, bacteriophages. The bacteriophages may cause disease as new antigens arriving with new bacteria, or be revealed as new antigens following induction in lysogenic organisms within the normal bacterial flora.
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