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Graham A. W. Rook Angus Dalgleish Infection, immunoregulation, and cancer Authors’ addresses Graham A. W. Rook1, Angus Dalgleish2 1 Department of Infection, University College London (UCL), London, UK. 2 St George’s Healthcare NHS Trust, London, UK. Summary: As man has moved rapidly from the hunter–gatherer environment to the living conditions of the industrialized countries, the incidences of some cancers have increased alarmingly. Recent increases are usually attributed to dietary changes or to altered exposures to putative carcinogens associated with the modern lifestyle. However, the changes in cancer incidence parallel similar increases in non-neoplastic chronic inflammatory disorders (inflammatory bowel disease, allergies, and autoimmunity), and the epidemiology is often strikingly similar. This parallel is worth exploring, because the increases in chronic inflammatory disorders are at least partly explained by immunoregulatory defects resulting from diminished exposure to microorganisms that co-evolved with mammals and developed a role in driving immunoregulatory circuits (the hygiene hypothesis). Dysregulated chronic inflammation can drive oncogenesis and also provides growth and angiogenic factors that enhance the subsequent proliferation and spread of tumor cells. Thus, a modern failure to downregulate inappropriate inflammation could underlie increases in some cancers in parallel with the increases in chronic inflammatory disorders. This possibility is supported by recent work showing that in some circumstances regulatory T cells protect against cancer, rather than aggravating it, as previously assumed. A greater understanding of these interactions might pave the way to improved microbe-based immunotherapies. Correspondence to: Graham A. W. Rook Department of Infection University College London (UCL) 46 Cleveland Street London W1T 4JF, UK Tel.: +44 20 7679 9489 Fax: +44 20 7607 0016 e-mail: [email protected] Acknowledgements The authors declare no conflicts of interest. Keywords: cancer, immunoregulation, Treg, hygiene hypothesis, infection, IL-10, TGF-b Introduction Immunological Reviews 2011 Vol. 240: 141–159 Printed in Singapore. All rights reserved 2011 John Wiley & Sons A/S Immunological Reviews 0105-2896 As man has moved rapidly from the hunter–gatherer environment to the living conditions of the rich industrialized countries, the incidences of some cancers have increased alarmingly. Some cancers (< 20%) are attributed to oncogenic changes or chronic inflammation caused by infections, but most of the recent increases have been attributed by oncologists to dietary changes or to altered exposures to putative carcinogens associated with the modern lifestyle. Interestingly, the changes in cancer incidence have been paralleled by even larger increases in a number of non-neoplastic chronic inflammatory disorders (inflammatory bowel disease, allergies, and autoimmunity), and the details of the epidemiology are often strikingly similar. This parallel is worth exploring, because chronic inflammation can contribute to oncogenesis 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 141 Rook & Dalgleish Æ Infection, immunoregulation, and cancer and often provides growth and angiogenic factors which enhance the proliferation and spread of tumor cells. A failure to regulate inflammation could underlie not only the chronic inflammatory disorders but also some cancers. If this hypothetical parallel were found to be real, it would provide important pathogenetic clues, because a significant part of the increase in chronic inflammatory disorders is now thought to be attributable to diminished exposure to a range of microorganisms and parasites that either co-evolved with humans or accompanied humans from at least as far back as the Paleolithic era. These organisms have been present throughout human evolution as commensals (notably in the intestinal microbiota), environmental ‘pseudocommensals’, causative agents of subclinical infections or asymptomatic carrier states, and helminth infections. Modern revisions of the ‘Hygiene’ or ‘Old Friends’ hypothesis suggest that such organisms have been tasked by co-evolutionary processes with establishing the ‘normal’ levels of immunoregulation within the immune system. Therefore, when exposure to the Old Friends is reduced, there is a relative failure to terminate inappropriate inflammatory responses, leading to increases in the chronic inflammatory disorders listed above (1). Here, in view of the crucial roles of inflammation in cancer, we explore the potential relevance of this concept to the changing incidences of several cancers in developed countries. Infection as a cause of cancer There have been numerous excellent reviews of the role of infections as causes of cancer (2–6). In contrast, there has been much less discussion of the possible roles of infections in the prevention of cancer, and in this review we seek to explore both aspects in parallel. Table 1 illustrates most of the organisms associated with cancer development, classified as far as possible according to how cancer is promoted (though there is much overlap). One purpose of the table is to demonstrate that inflammation is a crucial aspect of oncogenesis attributable to infection, while very few organisms cause cancer by direct infection and transformation of target cells. The lymphotropic oncogenic viruses Epstein–Barr virus (EBV), human herpesvirus 8 (HHV8), and human T-lymphotropic virus 1 (HTLV-1), are examples of the latter, and these viruses directly infect a subset of lymphoid cells, which they can then transform either directly or indirectly by transactivation of oncogenes, as in the case of HTLV-1. Little is known about the role in cancer causation of the truly intracellular infections that form the major focus of this Table 1. The major infectious agents that trigger cancer Mechanism Infect and transform lymphoid cells Transformation Inflammation and partial integration Chronic inflammation and oncogenic proteins Chronic stimulation of lymphocytes by pathogen antigens and ⁄ or autoantigens Chronic inflammation Chronic inflammation; possible associations Immunosuppression * Organism Cancer Epstein–Barr virus (EBV) Human Herpesvirus 8 (HHV8) Human T-lymphotropic virus 1 (HTLV-1) HPV HBV Hepatitis C virus (HCV) Helicobacter pylori Campylobacter jejuni Borrelia burgdorferi Chlamydia psittaci Hepatitis C virus (HCV) Bacteroides fragilis (enterotoxigenic) Malaria (cofactor) Tuberculosis Schistosomiasis Liver flukes Salmonella spp. Helicobacter pylori Propionibacterium acnes? Mycoplasma spp. ? ?? unknown prostatitis ?? HIV References * Burkitt’s lymphoma Kaposi sarcoma T-cell leukemia Cervical cancer Hepatocellular carcinoma Hepatocellular carcinoma Gastric lymphoma Immunoproliferative small intestinal dis. Cutaneous lymphoma Ocular lymphoma Spleen lymphoma Colorectal cancer Burkitt’s lymphoma Lung cancer Bladder cancer Cholangiocarcinoma Gallbladder cancer Gastric and esophageal adenocarcinoma Prostate Prostate Prostate EBV+ CNS lymphomas HHV8+ sarcoma (Kaposi) HPV+ ano-genital cancers (4) (2) (2) (4) (3) (29) (3) (30) (3) (146, 147) (7) (9, 10) (2, 5) (5) (8) (113, 115) (148) (149) (4) EBV also associated with nasopharyngeal carcinoma, Hodgkin lymphoma and non-Hodgkin lymphoma. C. jejuni produce CdtB (cytolethal distending toxin B) that causes direct DNA damage. HCV also encodes oncogenic proteins. 142 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 Rook & Dalgleish Æ Infection, immunoregulation, and cancer volume, though there are exceptions. Malaria can act as a cofactor for EBV-induced oncogenesis, because Plasmodium falciparum antigens such as PfEMP1 can induce reactivation of EBV (7). The increased viral load and the concomitant polyclonal B-cell activation with enhanced B-cell survival may augment the risk of endemic Burkitt’s lymphoma in children living in malaria-endemic areas (7). Similarly, although gallbladder cancer is relatively rare, the highest incidences are seen in populations where chronic infections like Salmonella typhi and S. paratyphi are prevalent, though obesity and multiparity are also consistently associated (8). More controversially, it has been reported that autopsies of patients with lung cancer frequently reveal the simultaneous presence of tuberculous lesions in the lungs (9). This observation might be meaningful, because it has been reported that there is an increased risk of lung cancer following a diagnosis of TB among farmers in Xuanwei, China. The risk was greatest within 5 years of diagnosis but remained elevated (approximately threefold) for more than 10 years. This association was seen in men and women and was not due to confounding effects of tobacco use or indoor environmental smoke (10). Nevertheless, the situation is likely to be more complex, because M. tuberculosis has immunoregulatory effects that differ according to whether latent or progressive infection has developed, and this is discussed later. In view of the paucity of direct evidence for involvement of intracellular infections in either preventing or causing oncogenesis (excluding viruses which are outside the remit of this article), certain other organisms will be used to illustrate their potential role. In particular, the involvement of Helicobacter pylori in gastric lymphoma and gastric and esophageal adenocarcinoma is of striking interest. Most H. pylori localize within the gastric mucus layer and do not directly interact with host cells. However, some organisms adhere to gastric epithelial cells and occasionally are internalized by these cells. Using fluorescent in situ hybridization (FISH) with probes for H. pylori 16S rRNA and cagA, H. pylori was detected inside metaplastic, dysplastic, and neoplastic epithelial cells (11). These findings are compatible with the view that an intracellular location is involved in the carcinogenic effect and fits with the observation that therapy aimed at eliminating H. pylori can induce regression of precancerous lesions (11). Chronic inflammation Inflammation is present in tumors and is fundamental to their development and spread. Inflammation can enhance mutation (12), growth, vascularization (13), and metastasis (Fig. 1). 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 Fig. 1. Some potential roles of infection in preventing and causing cancer (excluding direct transformation, which is discussed briefly in the main text). Epidemiological studies indicate that transient infection may provide some protection against cancer. However, chronic infection leading to chronic inflammation can initiate oncogenesis, and also, by subsequently providing growth and angiogenic factors, potentiate existing cancers. Tumor necrosis factor-a (TNF-a) may be particularly important (14), and it has been found to be secreted by a wide variety of tumor cells, including B and T-cell lymphoma, myeloid and lymphoid leukemias, carcinomas of the breast, colon, lung, ovary, pancreas, prostate, cervix, and glioblastoma, and neuroblastoma (reviewed in 15). There have been excellent recent reviews of these issues, which will therefore only be outlined briefly here (14–18). Background: inflammation in cancer First, tumors contain ‘smoldering’ inflammation (including release of TNF-a, as discussed above) that is not obviously related to any external inflammatory stimulus (17). This is partly because several important oncogenes such as RAS and MYC activate signaling pathways involved in inflammation (17). In addition, commonly seen mutations in tumor-suppressor genes such as Von Hippel-Lindau tumor suppressor (VHL), transforming growth factor-b (TGF-b), and phosphatase and tensin homologue (PTEN), can all lead to increased activation of transcription factors involved in inflammation and vascularization, particularly nuclear factor-jB (NF-jB), hypoxia inducible factor 1a (HIF-1a), and signal transducer and activator of transcription 3 (STAT3) (19). Moreover, many carcinomas secrete factors that upregulate fibronectin and recruit vascular endothelial growth factor receptor 1 (VEGFR1)-positive hematopoietic progenitors and inflammatory 143 Rook & Dalgleish Æ Infection, immunoregulation, and cancer cells to sites of tumor growth. These cells may also secrete factors that directly activate recruited myeloid cells to produce tumor-promoting cytokines such as TNF-a and IL-6 (14, 20). Increased rates of DNA damage and defective repair The mutation rate in inflamed tissues is higher than in normal tissues, though lower than in tumors (21). A number of mechanisms and pathways have been implicated in the increased mutation rate. Growth factors, cytokines, and chemokines produced by inflammatory cells and by the tumor itself induce increased expression of transcription factors such as c-Myc and also increase the activity of NF-jB, HIF-1a, and STAT3. These transcription factors alter the expression of hundreds of target genes related to cell growth, apoptosis, invasion, and inflammation. These processes accelerate the intrinsic mutation rate in cancer cells, at least partly by increasing levels of reactive oxygen species (ROS) and nitrogen species. ROS released from inflammatory cells can cause double strand breaks in the DNA of cancer cells. Moreover nitric oxide (NO) and the inflammatory cytokine interleukin-6 (IL6) increase the activity of DNA methyltransferase, resulting in extensive methylation of cytosine bases including those in CpG islands that can be located in promoters. This can lead to the silencing of promoters and loss of expression of the genes encoding essential mismatch-repair genes, such as hMLH1. This epigenetic change leads in turn to faulty DNA repair, resulting in microsatellite insertions and deletions within the coding regions of tumor suppressor genes. (microsatellites are normal, common, repeated sequences of DNA from 1to 6 bp, often located in promoter regions). The consequent microsatellite instability in tumor suppressor genes can lead to uncontrolled cell division and tumor growth (22). Among the genes that contain microsatellites in their coding regions are those encoding the TGF-b receptor 2, insulin-like growth factor 2 receptor, and BCL2-associated X protein (BAX). Another intriguing mechanism of DNA damage is ectopic expression of activation-induced cytidine deaminase (AID). This DNA ⁄ RNA editing enzyme normally induces hypermutation of the immunoglobulin loci in B cells. However, it can be ectopically induced in tumors by a variety of inflammatory cytokines. When ectopically expressed, rather than targeting immunoglobulin loci in B cells, AID produces mutations and translocations through induction of double strand breaks in the DNA of other cell types. Ultimately chromosomal instability results in abnormal segregation of chromosomes and aneuploidy (12). 144 Angiogenesis and metastasis In healthy adults, the growth of new vessels is largely confined to the female genital tract and to sites of wound repair. It is regulated by a balance between angiogenic factors such as vascular endothelial growth factor A (VEGFA), fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), and inhibitors of angiogenesis such as thrombospondin 1, angiostatin, endostatin, and tumstatin. The inflammatory cascades in tumors (Fig. 1), whether the intrinsic effects of the oncogenic mutations in the tumor cells themselves (17), secondary release of mediators from recruited inflammatory cells (20), or inflammation due to infection (23), can all lead to an imbalance, where inappropriate angiogenesis is encouraged and metastasis is facilitated (24–26). In a classic experiment, fibrosarcoma cells showed enhanced metastasis to the lungs in the presence of exogenous TNF, while neutralization of endogenous TNF led to a significant decrease in the number of pulmonary metastases (26, 27). Angiogenesis is further promoted by infiltration of monocytes expressing tyrosine kinase with immunoglobulinlike and EGF-like domains 2 (TIE-2). These TIE-2-expressing monocytes (TEMs) constitute a small pro-angiogenic monocyte subset that circulates in the mouse and human peripheral blood. TIE-2 is a cell-surface receptor for angiopoietins, and these cells are preferentially recruited to tumors and other sites of angiogenesis (18). Additional oncogenic effects of infection All the mechanisms described in the previous section can occur in tumors in the absence of infection. In the current context, the important point is that infections induce similar changes and so can initiate and promote tumors. Moreover infections can encourage oncogenesis by additional mechanisms described in this section. Infection-induced DNA abnormalities The immune response to infection can lead to local release of ROS and nitrogen species, thereby increasing the likelihood of oncogenic mutations. Interestingly, methylation changes affecting CpG islands in promoters of tumor suppressor genes like those discussed above are also found in a model of Helicobacter infection (28). Chronic stimulation of lymphoid cells Chronic stimulation by microbial antigens or autoantigens is associated with a distinct subset of extranodal lymphomas 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 Rook & Dalgleish Æ Infection, immunoregulation, and cancer arising from B cells of the marginal zone of mucosa-associated lymphoid tissue (MALT) or of the spleen (3, 29, 30). The implicated organisms include H. pylori, Campylobacter jejuni, Borrelia burgdorferi, Chlamydia psittaci, and hepatitis C virus (HCV). For example, H. pylori-infected gastric mucosal cells produce proinflammatory cytokines (such as lymphotoxin beta) and B-cell homing factors (such as BCA-1), leading to the emergence of MALT in the gastric mucosa. While some of the chronic stimulation is likely to be attributable to ongoing responses to microbial antigens, in several cases it seems that the major stimulus comes from an autoimmune response triggered by the infection. For example, neoplastic B cells from gastric MALT lymphoma are not specific for H. pylori antigens but rather for autoantigens found in the gastric mucosa (3). These autoreactive B cells are thought to receive cognate help from H. pylori-specific T cells displaying crossreactivity with gastric autoantigens (31, 32). Indeed autoimmunity, even when not directly attributable to a known infection (e.g. Sjögren’s syndrome) is associated with an increased risk of lymphomas. Thus, several mechanisms may account for the finding that in at least 60% of patients, eradication of H. pylori can cause regression of low-grade MALT lymphoma, and of early stage high-grade lymphomas of the stomach (33). These mechanisms include reduction in bacterial antigen load, removal of an ‘adjuvant’ stimulus for autoimmunity, and elimination of abnormally functioning H. pylori-infected cells. Similar arguments apply to several of the other organisms implicated in the induction of extranodal lymphomas. C. jejuni is associated with Guillain–Barré syndrome, which is caused by cross-reactive antibodies that bind both to C. jejuni lipooligosaccharides and to gangliosides in the nervous system (34). Similarly, the OspA protein of B. burgdorferi is structurally homologous to human lymphocyte function antigen-1 (LFA1) and may play a role in the autoimmune manifestations and chronic activation of the immune system that leads occasionally to lymphoma (35). Damage, atrophy, metaplasia, dysplasia, and cancer A further mechanism is involved when chronic infections lead to chronic tissue damage. Detailed histopathological analysis reveals the sequence of events that occurs in H. pylori-induced adenocarcinoma of the stomach. First there is chronic active non-atrophic gastritis, which is followed by focal loss of glands (atrophy) particularly at the antrum-corpus junction, with fibrous stromal tissue replacing the lost glands. Next there is metaplasia, with the appearance of cells initially 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 resembling various types of differentiated small intestinal epithelial cells and later resembling colonic mucosa. These events might be an early phase of dysplasia. Dysplasia is characterized by atypical changes in nuclear morphology and tissue architecture. At some point, adenocarcinoma develops in these lesions. Some investigators believe that the initial stages of inflammation and atrophy create an abnormal microenvironment favoring engraftment of bone marrow-derived stem cells (BMDCs) that replace the depleted local tissue stem cells. These BMDCs are in a distinct epigenetic state and seem susceptible to mutation and deviation away from the pathway of complete differentiation, resulting in progressive loss of differentiation, uncontrolled replication, and eventual neoplastic invasive behavior (36). If this is correct, then infectioninduced chronic inflammation can cause cancer by attrition of local stem cells, and their replacement with more dysplasiasusceptible BMDCs. Infections may also reduce cancer In addition to contributing oncogenic effects in the ways described above, infections may also reduce the risk of cancer (Fig. 1). The mechanisms fall into three broad categories. First, infection might boost the host’s own immune response to cancers because of adjuvant and immunostimulatory effects. Second, cross-reactivity between microorganisms and tumor antigens may lead to the priming of anti-tumor effector cells. Third, infections can modulate immunoregulatory circuits, as recognized in relation to the Hygiene or Old Friends hypothesis. These three mechanisms are discussed below. It is not always possible to know which particular one is involved, and it is likely that many infections induce more than one of them. Adjuvant effects Although chronic infection, by causing chronic inflammation, is likely to drive oncogenesis, transient exposure to microbial components such as endotoxin (LPS) or transient infections would be expected to enhance immune responses (Fig. 1). In the mid-19th century, Coley (37) had noted remission of cancer in patients developing severe infections, and he attempted to use bacterial extracts as a therapy, with some evidence of success. This work inspired an epidemiological study of people developing melanomas. The study concluded that the risk of melanoma was inversely related to the numbers of their recorded infections, particularly when accompanied by high fevers (38). Patients with histories of one, two to three and four or more infections had odds ratios of 0.66, 0.63, and 145 Rook & Dalgleish Æ Infection, immunoregulation, and cancer 0.32 respectively, and the trend was statistically significant (P = 0.004) (38). LPS, like many microbial components, is a powerful adjuvant. This quality has been thought to explain why workers in the cotton industry in Shanghai have reduced incidences of several cancers. Their heavy exposure to LPS might be protective against cancers of the esophagus, stomach (39), breast (40), and lung (41). Similarly, dairy workers, who are exposed to a multitude of microbial components including LPS, have been shown to have lower incidence of lung cancer (42). In the 1970s, a number of authors claimed that BCG vaccination protected against leukemia and other hematological tumors, though the validity of the findings remains uncertain (43). To date, one can only speculate as to what effect latent tuberculosis, affecting one third of the world’s population, has on the incidence of cancer. Cross-reactivity It appears that glycosylation changes are early consequences of oncogenesis, and glycoproteins on tumor cells bear oligosaccharides that are markedly different from those found on proteins produced by normal cells (44). It is suggested that these may sometimes resemble the oligosaccharide structures of bacteria (45), with more exposed galactose, N-acetylgalactosamine, and mannose (Fig. 1). This may go some way to explain why bacterial infections may be protective in cancer, as they might prime immune responses to cross-reactive epitopes on tumor cells. Another documented cross-reactive antigen is the HERVK-MEL epitope expressed in many melanomas. It is encoded by a human endogenous retrovirus of the HERV-K family. Sequences predicted to cross-react with this epitope were identified in BCG, vaccinia virus used for the smallpox vaccine, and the 17D yellow fever vaccine, all of which appear to afford some protection against development of melanoma (45–47). Modulation of immunoregulatory circuits It was pointed out above that dairy workers might be protected from ⁄ have lower risk of developing lung cancer (42). An adjuvant effect of LPS is a possible explanation, but others may be more relevant. Exposure to cowsheds also protects from allergic disorders (48), and this observation forms one of the strongest epidemiological findings underpinning the Hygiene hypothesis, outlined in the Introduction. One might speculate that the ‘cowshed’ effect downregulates Th2 responses, terminates inappropriate chronic (potentially 146 oncogenic) inflammation, and so simultaneously reduces oncogenesis, and improves the potential for Th1-dominated anti-cancer responses. The implications of the Hygiene hypothesis for cancer are expanded in the next section. Hygiene hypothesis The Hygiene hypothesis seeks to explain the rapid simultaneous increases in the incidences of several types of chronic inflammatory disease in the rich developed countries. These include autoimmunity [e.g. multiple sclerosis (MS) and Type 1 diabetes] and inflammatory bowel diseases (IBDs) (e.g. Crohn’s disease and ulcerative colitis) (49). All of these chronic inflammatory disorders show evidence of defective immunoregulation. Regulatory defects have been found in individuals suffering from allergic disorders (50), some autoimmune diseases (51, 52), and probably in IBD too (53, 54). This suggests that in developed countries there is a generalized defect in regulation of the immune system that is manifested in different individuals as a failure to terminate inappropriate responses to allergens (allergy), self (autoimmunity such as MS or Type 1 diabetes), or gut contents (IBD). This can be explained by an environmental effect operating at the population level, with superimposed genetic effects operating at the level of the individual (Fig. 2). We know that a generalized dysfunction of immunoregulatory mechanisms can lead to simultaneous increases in these diverse types of pathology, because genetic defects of forkhead box protein 3 (Foxp3), a transcription factor that plays a crucial role in the development and function of regulatory T cells (Tregs), leads to a syndrome known as X-linked autoimmunity–allergic dysregulation syndrome that includes aspects of allergy, autoimmunity, and enteropathy (55). A recent reformulation of the Hygiene hypothesis (Old Friends Hypothesis) suggests that certain organisms that were constantly present throughout mammalian evolutionary history had to be tolerated and so co-evolved a role in driving regulatory pathways within the immune system. The depletion of these organisms from the modern environment is thought to be contributing to the increase in immunoregulatory disorders (1). The organisms involved in the Hygiene hypothesis Common infections of childhood do not protect against allergies (56–58), Type 1 diabetes (59), or IBD (60–62). Rather epidemiological, experimental, and clinical studies have indicated that the important organisms in this context are relatively benign organisms such as lactobacilli, helminths, orofecally transmitted infections inducing carrier states, and 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 Rook & Dalgleish Æ Infection, immunoregulation, and cancer Fig. 2. The Hygiene or Old Friends hypothesis. The organisms with which mammals co-evolved (see Fig. 3) had to be tolerated, and mammals are now in state of evolved dependence on their ability to drive immunoregulatory pathways. In developed countries, immunoregulation is therefore decreased, and gene–environment interactions cause a subset of people living in developed countries to develop chronic inflammatory disorders. The nature of the disorder (if any) depends on the genetic background of the individual. Table 2. Some organisms implicated in the ‘Old Friends’ hypothesis Organism References Hepatitis A virus Helminths Helicobacter pylori Salmonella Gut microbiota Mycobacterium tuberculosis Toxoplasma gondii (unknown ? microbial)* (150, 151) (152, 153) (117) (154) (123, 155) (156, 157) (57, 151, 158) (48, 159) * Protection from cowsheds and homes contaminated by animal feces. saprophytes from mud and untreated water (1). All have long-standing associations with humans but have become depleted by modern lifestyles. These organisms are listed and referenced in Table 2. They were all present from very early in human evolution and became depleted during the second epidemiological transition: during industrialization, the construction of modern cities, concrete, tarmac roads, and separation from mud animals and feces that had always previously provided contact with them (Fig. 3). Pro-inflammatory effects of previously beneficial genetic factors The depletion of regulation-inducing organisms leads to a further complication. In the developed countries, genetic factors that were not previously causing disease can assume negative 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 roles when the Old Friends are removed. In areas with the highest burdens of immunoregulation-inducing organisms, accumulation of compensatory single nucleotide polymorphisms (SNPs) or other genetic variants tended to increase release of pro-inflammatory cytokines (63–65) or enabled enhanced Th2 responses to help control parasite burdens (66). However, as soon as the immunoregulatory organisms (Figs 2 and 3) are removed, these SNPs lead to exaggerated responses, and become risk factors for chronic inflammatory disorders (Fig. 4). Hygiene hypothesis and cancer As summarized above, the Hygiene hypothesis suggests that the modern lifestyle is leaving the immune system with inadequate immunoregulatory mechanisms, causing, in some individuals, increased susceptibility to a variety of chronic inflammatory disorders. As many cancers are increasing in incidence too, it is interesting to speculate about a possible connection. It is usually assumed that cancer is associated with excessive immunoregulation rather than too little, because many experimental systems have shown detrimental effects of Tregs in established tumors. However in view of the clear role of inflammation in oncogenesis, there is the obvious possibility that mechanisms described by the Old Friends Hypothesis may be protective by terminating inappropriate inflammation, 147 Rook & Dalgleish Æ Infection, immunoregulation, and cancer Fig. 3. The Darwinian background to the Hygiene or Old Friends hypothesis. The organisms implicated by epidemiology, clinical studies, in vitro experiments, and animal models are shown highlighted in bold font on the right. These organisms accompanied man at least as far back as the Paleolithic era and were part of mammalian evolutionary history. They were still present, often in larger quantities, after the first epidemiological transition (Neolithic). Epidemiological studies indicate that the new sporadic infections picked up from domesticated animals following animal husbandry in the Neolithic are not so relevant. The important organisms were progressively lost from the start of the second epidemiological transition in the early 19th century. Factors not usually sufficient to cause disease present from the paleolithic on their role in induction of immunoregulation Genetics Environmental triggers Compensatory SNP or alleles 1) pro-inflammatory cytokines 2) IgE Delayed exposure to viruses ? Diet, obesity progressively from mid 18th C as contact with soil, animals and faeces diminished Potential susceptibility to Th1/ Th17 or Th2 disorders in the Vit D deficiency, Pollutants (dioxins) etc Allergies, MS, IBD, type 1 diabetes etc and some cancers? Fig. 4. The additional effects of compensatory single nucleotide polymorphisms (SNPs) and of aggravating environmental changes. In parts of the world with high levels of immunoregulatory Old Friends, there has been selection for compensatory SNPs that restores the inflammatory response. This has established an equilibrium that is disturbed when the Old Friends are lost during the second epidemiological transition. The same SNPs then become risk factors for chronic inflammatory disorders. This situation is likely to be aggravated by other modern environmental changes that also tend to increase inflammatory responses, such as obesity, vitamin D deficiency, and pollutants such as dioxins that drive Th17 cells. which is a contributing factor to the initiation of some cancers and to the accelerated growth of others (Fig. 1). It might be very significant that non-steroidal anti-inflammatory agents, 148 such as cycloxygenase-2 (COX-2) inhibitors, reduce both the risk of developing certain cancers (such as colon and breast cancer) and the mortality caused by these same cancers (67). 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 Rook & Dalgleish Æ Infection, immunoregulation, and cancer This suggestion would be strengthened if there were evidence that anti-inflammatory cytokines of the types associated with immunoregulatory pathways were found to be protective. Effects of IL-10 on cancer In IL-10) ⁄ ) mice, severe inflammation occurs in the gut as soon as colonization with normal microbiota occurs. This inflammation is mediated by CD4+ Th1-like cells and activated macrophages and is attenuated by administering antiIFN-c antibody. Eventually, the inflammatory lesions resemble early adenocarcinomas. Restoration of IL-10 is protective: IL-10) ⁄ ) mice treated with exogenous IL-10 daily from 3 weeks of age remained free of disease. Similarly, administration of IL-10 prevented the colitis that occurs in 100% of SCID mice transplanted with CD45RBhi CD4+ effector T cells. Interestingly, administration of IL-10 to adult IL-10) ⁄ ) mice after the colitis was established did not cure the colitis but it still very significantly reduced the incidence of adenocarcinomas (68). These results imply that in addition to the anti-inflammatory effect, IL-10 is also involved in other protective antitumor mechanisms, and further studies do indeed confirm this finding. First, there is strong evidence that IL-10 encourages NK cell-mediated tumor cell killing by downregulating MHC class I, modulating NK cells, and inhibiting angiogenesis (69–72). It has also been reported that systemic administration of IL-10 induced an effective specific immune response against established tumors in mice. IL-10-treated mice were immune to subsequent rechallenge with 10-fold larger numbers of the same but not a different tumor (73). This work in mice with laboratory tumor cell lines might be too artificial for extrapolation to humans, but it does imply that IL-10 encouraged an adaptive immune response. In conclusion, it is not surprising that the presence of high levels of IL-10 in the serum or within the tumor can be a favorable prognostic indicator for some tumors but an unfavorable one for others (72). Effects of TGF-b on cancer The effects of TGF-b on cancer remain controversial. Many tumors secrete TGF-b, a potent suppressor of the cytotoxic T-cell response, a crucial anti-tumor effector mechanism, thereby promoting tumor growth (74, 75). In addition, there is evidence to suggest that Tregs inhibit the anti-tumor functions of NK cells by expressing membrane-bound TGF-b (76). Moreover, TGF-bs are involved in epithelial to mesenchymal transdifferentiation. The switch of carcinoma cells to an invasive phenotype has some similarities to this normal process, and TGF-b can induce epithelial to mesenchymal 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 transition in vitro in numerous types of cancer cells (77). Similarly, control of angiogenesis is a normal function of TGF-b, partly via induction of VEGF, which explains why knockout of the genes encoding TGF-b1 or TGFbRII is lethal to embryos. It is therefore not surprising that neutralizing antibodies to TGF-b can reduce angiogenesis in tumors (77). Hence, there are several ways in which TGF-b might promote the growth of established tumors. TGF-b may, however, have a different role in tumor initiation. The broad anti-inflammatory effects of TGF-b might be expected to counter inflammation-induced oncogenesis described earlier. Similarly, TGF-b is a potent inhibitor of growth of epithelial cells and plays a role in the homoeostasis of healthy epithelial surfaces (77). Indeed mutations in human TGFBR2 and in SMAD2 and SMAD4, which are involved in transduction of signals from the activated TGF-b receptors, have been found in both sporadic and inherited colon cancer. Experiments in TGF-b) ⁄ ) mice cast some light on the mechanism. Germ-free TGF-b) ⁄ )RAG2) ⁄ ) mice do not develop colitis or cancer. However, when exposed to H. hepaticus, both colitis and cancer were observed (78, 79). Interestingly, RAG2) ⁄ ) mice, which retain the ability to secrete TGF-b, remain cancer-free even in the presence of H. hepaticus, indicating that TGF-b can block oncogenic effects of the H. hepaticus-induced inflammation. In conclusion, the available data are compatible with the view that TGF-b may protect against the initiation of cancer via its effects on cellular homeostasis and its anti-inflammatory roles. In contrast, TGF-b might promote growth of tumors once these are established. Effects of Tregs on cancer There have been numerous reviews of the detrimental role of Tregs in progressive established cancer (75), and other reviews of the ways in which cancer therapy might be improved by drugs that oppose the functions or development of Tregs (80). However, since regulatory cytokines can be shown to have protective effects against the induction of cancers and, in the case of IL-10, some protective effects even after the tumor has formed, we should expect to find evidence that Tregs can protect against oncogenesis, perhaps particularly in relation to cancers that are known to be associated with uncontrolled chronic inflammation (Fig. 5). Beneficial role for Tregs in animal models of cancer An example of the protection from oncogenesis by Tregs was seen in C57BL ⁄ 6 mice infected with Helicobacter felis. This 149 Rook & Dalgleish Æ Infection, immunoregulation, and cancer Beneficial roles of Treg Treg block oncogenesis in site of chronic inflammation. Treg in tumour bed reduce inflammation, and so block release of growth and angiogenic factors. IL-10: Anti-inflammatory Increase NK activity Inhibit angiogenesis TGF-β β: Anti-inflammatory Inhibit epithelial growth Treg from animals that have experienced infection Detrimental roles of Treg Treg in tumour stroma or local lymphoid aggregates block development of protective cytotoxic lymphocyte (CTL) response. IL-10: Inhibit protective anti-tumour response TGF-β β: Inhibit CTL Treg from ‘hygienic’ animals Fig. 5. The heterogeneous roles of Tregs and anti-inflammatory cytokines in cancer. It is well documented that Tregs can oppose protective responses to cancer. It is now emerging that Tregs of certain poorly defined phenotypes, in certain locations, can be protective against both initiation and progression of cancer. Tregs can terminate chronic inflammation and so stop oncogenesis. In existing tumors, Tregs can block release of growth and angiogenic factors, but to achieve this, they need to be in the tumor bed itself. Tregs located in surrounding lymphoid aggregates are more likely to inhibit generation of the protective T-cell response. We still know little about the control of the localization or phenotype of these Tregs, but in animal models (see Fig. 6), Tregs from infected animals can have the relevant properties, whereas Treg from very clean specific-pathogen free (SPF) animals do not. organism induces Th1- and IFN-c-dependent gastric atrophy, a premalignant lesion reminiscent of that which can occur in humans infected with H. pylori. However, when H. felisinfected mice were co-infected with Heligmosomoides polygyrus, a natural murine nematode parasite with a strictly enteric life cycle, there was minimal gastritis, and the premalignant atrophic lesions were not seen (81). The co-infection did not eliminate inflammation. Indeed, 16 weeks after infection gastric colonization with H. felis was greater in the group coinfected with H. polygyrus, particularly in the antrum, with extension into the corpus mucosa. However, the inflammation in the co-infected mice was quite different, with strikingly reduced IFN-c and increased expression of IL-10, TGF-b, and some Th2 cytokines (81). A similar effect was revealed using Helicobacter hepaticus, another natural enteric bacterial pathogen of mice. 129 ⁄ SvEv RAG-2-deficient mice were infected with H. hepaticus by gastric gavage. Many of these animals develop IBD and colon cancer. However, when the H. hepaticus-infected mice were treated at 1, 3, or 12 months after infection with adoptive transfer of CD4+CD45RBloCD25+ regulatory cells, they had reduced severity of IBD and significantly lower risk of colon cancer (82), but only if the Tregs came from IL-10-competent donors. When Tregs from IL-10) ⁄ ) mice were used, there was an increased incidence of mucinous tumors (82). Thus, these models show that induction of Tregs by co-infection with a helminth (a mechanism discussed above in relation to the Hygiene hypothesis) or passive transfer of Tregs can convert an inflammatory 150 response from a pattern that causes cancer to one that does not. The same group also investigated the APCMin ⁄ + mouse. APC (adenomatosis polyposis coli) is a human gene that causes colorectal cancer when it is mutated. APCMin mice have a point mutation in the murine homologue, and they develop multiple intestinal adenomas and mammary tumors. However, the number of epithelial adenomas is reduced when heterozygote APCMin ⁄ + mice are treated with CD4+CD25+ Tregs, but as in the H. felis model discussed above, only if the Tregs come from IL-10-competent donors (83). Further work has confirmed the requirement for IL-10 but also revealed that not all Tregs have the same ability to inhibit oncogenesis (Fig. 6). Tregs transferred from donors that had been preinfected with H. hepaticus protected against development of cancer in several models (23). The models were: (i) colorectal cancer (CRC) developing from inflammatory bowel disease in H. hepaticus-infected RAG2) ⁄ ) 129 ⁄ SvEv mice; (ii) CRC and mammary adenocarcinoma in H. hepaticus-free APCMin ⁄ + mice given pro-inflammatory effector T cells; and (iii) spontaneous mammary adenocarcinoma in FBV Her2 ⁄ Neu mice. In each case, even when no Helicobacter was present in the recipient (i.e. the cancer was spontaneous, or triggered by transfer of pro-inflammatory T cells), only Tregs from Helicobacter-experienced mice were protective (Fig. 6). When derived from H. hepaticusnaive ‘hygienic’ animals, they exacerbated the cancer in the first two models and had no effect in the third one. The 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 Rook & Dalgleish Æ Infection, immunoregulation, and cancer Treg-deficient effector T cells (CD4+CD45RBhi) Mammary adenocarcinoma C57Bl APCmin+/– 129 Rag2–/– Infect with Helicobacter hepaticus Transfer of Treg from infected mice is protective Colorectal cancer Fig. 6. Examples of beneficial effects of Tregs in animal models of cancer. Transfer of Tregs from animals infected with Helicobacter hepaticus into animals developing the cancers was protective, even when this organism was not involved in the cancer concerned and was not present in the recipient. By contrast, Tregs from specific pathogen-free animals were not protective, indicating that the precise phenotype of the Treg is crucial. exacerbation might have been due to inadequate release of IL-10, to conversion to Th17, or to a change in homing. This implies a functional difference in the Tregs, independent of their specificity. In conclusion, this series of experiments has shown that a gut-associated organism (H. hepaticus) can induce and modulate Tregs in ways that endow them with the ability to control cancer-promoting inflammation throughout the body (i.e. in the breast as well as the gut) via an IL-10-dependent mechanism. This constitutes experimental evidence for the relevance of the Hygiene hypothesis to cancer. Location and function of Tregs in cancer The strategies that are effectively anti-tumor in the models discussed in the previous section also caused a change in the location of Foxp3+ Treg cells. Tumor progression was associated with Foxp3+ cells in lymph nodes and in tissue surrounding the tumor, whereas remission was associated with Foxp3+ cells in the tumor itself (23). Female FVB strain MMTV-HER2 ⁄ neu (ErbB2) transgenic mice have a genetically determined tendency to develop mammary tumors resembling those seen in 30% of humans with breast cancer. Growth of the tumors requires ongoing inflammation. Working with mice already bearing tumors, it was found that administration of anti-TNF significantly decreased subsequent tumor growth. Interestingly, the anti-TNF also caused accumulation of Foxp3+ cells in the tumor itself, whereas in untreated animals with more rapidly progressing tumors, there were no Foxp3+ cells in the tumor but instead an accumulation in the lymph nodes and tissue surrounding the tumor. These authors observed a similar phenomenon in the APCMin ⁄ + mouse model (23). 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 This observation might be relevant to human disease. The assessment of numbers of circulating Tregs or of Tregs infiltrating the tumor itself has been used as a prognostic indicator in human cancers, though the correlations found depend on the tumor studied. Their presence has been claimed to be a negative prognostic indicator in carcinomas of the lung, pancreas, stomach, liver, and ovary, whereas Tregs appeared beneficial in B-cell lymphoma, head and neck, and colon carcinoma, though in other studies numbers of Tregs have not correlated with changes in survival (reviewed in 84). This heterogeneity of results suggests that something is being missed, and the animal models discussed above suggest that the localization of the Tregs might be crucial. A study that counted infiltrating Tregs in breast cancer concluded that high numbers of these cells were detrimental to survival (85), but a further study that recorded the precise location of the Tregs concluded that the presence of Foxp3+ Tregs within the tumor bed did not affect the patients’ clinical evolution, whereas their presence in lymphoid-enriched areas correlated with a higher risk of relapse, a shorter relapse-free survival, and reduced overall survival (84). This finding agrees with the animal models described above (23) and might suggest that Tregs in the lymphoid aggregates can inhibit the development of effector cells such as CTLs, whereas Tregs in the tumor bed can usefully inhibit the tumor-promoting effects of inflammation discussed earlier. The microanatomical location is also crucial when considering the role of tumor-infiltrating macrophages. These correlate with improved survival if present in the tumor ‘islets’, among the tumor cells, but are linked to a poor prognosis if present in the surrounding stromal tissue (86). It is possible that stromal macrophages support tumor growth by enhancing formation of tumor stroma and angiogenesis, whereas macrophages in contact with tumor cells are cytotoxic, and so limit tumor growth. As a further example, in a murine model of colon adenomatous polyposis, there is focal mastocytosis that promotes tumor initiation and progression (87, 88). Adoptively transferred Tregs suppress this mastocytosis. In contrast, the endogenous Tregs that are present in the polyps in large numbers show proinflammatory activity and promote differentiation and expansion of mast cells (87, 88). It is known that Tregs can be incapacitated by exposure to TNF (89) or IL-6 (90), so it may be that when Tregs fail to suppress inflammation, perhaps because TNF and IL-6 are being released spontaneously from the tumor cells (and in this model, by the mast cells) their function can be perverted. Indeed, Treg cells that expand in adenomatous polyps no longer produce IL-10 and 151 Rook & Dalgleish Æ Infection, immunoregulation, and cancer instead switch to production of the pro-inflammatory cytokine IL-17 (87). Treg modulation of inappropriate inflammatory mechanisms in cancer Tregs not only inhibit inflammatory responses but also influence the balance between different effector cell types and selectively alter cytokine secretion profiles. For instance, Foxp3+ Tregs can functionally differentiate to acquire an ability to specifically control Th1 or Th2 type immune responses by modifying their expression of Th1-related or Th2-related transcription factors (91). Thus, some natural Foxp3+ Tregs express T-bet, which increases when they are stimulated in a Th1 cytokine environment. This causes upregulation of C–X– C chemokine receptor 3 and promotes recruitment to sites of type 1 inflammation. Foxp3 also controls expression of the transcription factor IFN regulatory factor 4 (IRF4), which is required for Th2 differentiation. Deletion of IRF4 from Tregs impairs their ability to control Th2- but not Th1-mediated inflammation (92). Therefore not only is the location of the Treg crucial but also their precise function. They need to be able to inhibit the types of T cell that are detrimental to tumor immunity such as Th2 cells. The inflammatory cells that infiltrate tumors are often not well adapted to the role of driving Th1- and CTL-mediated anti-tumor responses and show a Th2 bias. The dendritic cells tend to have an immature phenotype, and the myelomonocytic cells express the alternative M2 phenotype (19). M2 macrophages are driven by signals such as IL-4, IL-13, glucocorticoids, and IL-10. They express low IL-12 but high IL-10, IL-1decoyR, IL-1ra, and scavenger receptors (SR-A and mannose receptors). These cells promote Th2 responses, scavenge debris, and drive angiogenesis and tissue remodeling as discussed earlier (18). Thus although there is strong evidence that Th1-mediated inflammation has a particular tendency to initiate oncogenesis (68), once a tumor is established, a switch to a Th2 pattern may reduce anti-tumor mechanisms, while the Th2-associated repair and angiogenic functions facilitate growth (18, 19). In this context, it is interesting that several of the organisms involved in the Hygiene hypothesis have the ability to drive Tregs that can suppress Th2 responses. This includes M. vaccae (93), various helminths (94–96), and H. pylori (discussed in detail below). Epidemiology of cancer in relation to infection An examination of the epidemiology of some of the cancers that increase markedly in incidence as developing countries 152 adopt the lifestyle of rich industrialized countries reveals trends that mimic those that led to the invocation of the Hygiene hypothesis to explain the rising incidence of chronic inflammatory disorders. For instance, age-specific incidence rates for specific cancers in Asians correlate with the state of economic development of their country of residence. Incidences are much lower in Asians living in India compared to those living in the UK or USA (97). Hodgkin’s lymphoma The risk of classical Hodgkin’s lymphoma (HL) is inversely associated with socioeconomic status (98). In the West, HL is most prevalent among whites, followed by blacks and Hispanics, with the lowest incidence in Asians. Nevertheless, although incidence is low in Asian subgroups (presumably for genetic reasons), rates in Asians residing in the US are almost double the rates in Asians living in Asia, so there are clearly environmental factors that partly compensate for this resistance when Asians move to the USA (99). Early exposure to other children at nursery school and day care seems to decrease the risk of HL in young adults, and the risk is lowest among those with multiple older but not younger siblings. Having older siblings is associated with earlier exposure to common childhood pathogens and Old Friends (100), and this is precisely the observation that, in relation to allergies, led to the Hygiene hypothesis (101). We know that delayed exposure to some infections that are usually harmless in neonates can result in serious disease (e.g. polio or hepatitis A virus) and delayed exposure to viruses has also been considered by some authors in relation to the rise in Type 1 diabetes (102). There is evidence that HL is associated with EBV, which appears to increase the risk fourfold. Clearly the vast majority of individuals with EBV do not develop HL (100, 103), and this association does not explain the socioeconomic or family size data. Acute lymphatic leukemia of childhood The epidemiology of acute lymphatic leukemia (ALL) of childhood also shows striking parallels with the epidemiological findings that gave rise to the original version of the Hygiene hypothesis (101, 104). A study in Northern California provided preliminary evidence that protection from ALL is proportional to the number and frequency of social contacts (105). A large population-based case–control study [The UK Childhood Cancer Study (UKCCS)] revealed further evidence that social contacts in infancy can reduce risk of childhood ALL (106). Interestingly, early playgroup 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 Rook & Dalgleish Æ Infection, immunoregulation, and cancer Normal (ancient) Mid 19th–early 20th C Loss of helminths ------------> Gastric atrophy - B12 deficiency - Iron deficiency - Some adenocarcinoma in old age Late 20th C --- Loss of H. pylori (antibiotics) ---> Peptic ulcer disease shows sharp peak Sustained high acid, oesophageal adenocarcinoma Progressive loss of Treg, more allergic disorders Fig. 7. The changing role of Helicobacter pylori. Human studies and animal models suggest that in the presence of co-infection with Treg-inducing helminths, the inflammatory response induced by H. pylori is attenuated but involves the whole stomach. Adenocarcinoma is a late consequence in some individuals. As helminths are progressively eliminated, a more vigorous response occurs, and peptic ulceration is seen in individuals whose Treg response to H. pylori itself is suboptimal, while H. pylori becomes confined to the pylorus. More recently, the situation has been complicated by the fact that long-term infection with H. pylori results in decreased acid production. Thus, total loss of H. pylori due to antibiotics results in sustained high levels of acid production and consequent esophageal adenocarcinoma (modified and redrawn from reference 113). attendance appeared to give some protection against not only ALL but also to a limited extent against other pediatric cancers such as central nervous system tumors (106, UKCCS investigators, unpublished data quoted in 104). Equally striking is the fact that an analysis of data from 40 countries revealed a very significant positive correlation between incidences of ALL and Type 1 diabetes. Both diseases also correlated positively with GDP (107). A similar analysis had previously revealed a correlation between Type 1 diabetes and allergic disorders (108). Carcinoma of the stomach or esophagus The story of H. pylori is particularly instructive, because we know which organism is involved and we have some insight into the mechanisms. The majority of epidemiologic studies have reported a decreased incidence of gastric cancer in Africa compared to other countries where H. pylori infection is endemic. This is often described as ‘The African Enigma’. Looking at IgG subclasses as an approximate measure of the relative balance of Th1 and Th2-mediated response it was noted that the citizens of Soweto (an economically impoverished township near Cape Town, South Africa) had a more Th2-biased response to H. pylori than did people from Europe or Australia (discussed in 109). An appealing hypothesis is that the inflammatory effect of H. pylori in man is modified by simultaneous helminth infections, as demonstrated in experimental 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 models outlined earlier (23, 81). The milder chronic inflammation or increased immunoregulatory networks seen in the presence of helminths might mitigate the risk of stomach cancer (Fig. 7). Gastric ulcer disease due to H. pylori appeared in the 19th century in the USA and Europe, and its prevalence peaked in the early 20th century. Duodenal ulcer disease appeared and peaked 10–30 years later (110, 111). This again could be due to changes in immunoregulation; peptic ulcer disease is associated with reduced expression of Foxp3, IL-10, and TGF-b in gastric biopsies from H. pylori-infected donors (112). The modern lifestyle may be associated with poorly controlled Th1-orientated inflammation, causing more acute local damage and also causing the infection to be more frequently confined to the antrum of the stomach, whereas the typical situation observed in patients in developing countries is a milder pan-gastritis (113). Modern hygiene and antibiotics are leading to populations where many individuals do not carry H. pylori at all. Some authors believe that this total absence of colonization by H. pylori may be responsible for recent increases in the incidence of short- and long-segment Barrett’s esophagus and its malignant complications (114, 115). The reasoning here is that in the absence of the complex biological effects of H. pylori, which include mucosal atrophy, acid production by the stomach is excessive, leading to chronic inflammatory esophagitis and carcinogenesis (Fig. 7). 153 Rook & Dalgleish Æ Infection, immunoregulation, and cancer It is important to note that H. pylori is itself a potent inducer of Tregs, both locally in the stomach and duodenum (112, 116), and systemically (116), though Tregs are reduced in patients who develop ulcers (112). The changes in malignant and non-malignant manifestations of H. pylori described above have coincided with the striking increases in allergic disorders that prompted the Hygiene hypothesis. Interestingly, epidemiological studies indicate that infection with H. pylori protects against allergic disorders (57, 117), and similar effects are seen in laboratory models (118, 119). Thus H. pylori may provide an interesting example of the parallel between the hygiene of Old Friends hypothesis as applied to chronic inflammatory disorders and its application to cancer. Colon cancer Worldwide, colorectal cancer incidence rates increase as countries undergo the second epidemiological transition to economic development and remain high in longstanding, economically developed countries such as Japan, Australia, Western Europe, and North America (120). Part of this increase is associated with the increase in inflammatory bowel disease (IBD), in which the Hygiene hypothesis is already implicated. As outlined above, helminth infection can reduce colorectal cancer by inducing anti-inflammatory immunoregulation in a mouse model (81), and helminths have a therapeutic effect in human IBD (121, 122). However, other components of the microbiota also have important immunoregulatory functions (123). The microbiota can be altered by inappropriate diet, and obesity is associated with a distinct pattern of changes in the gut microbiota (124). Similarly, antibiotics have profound effects on the nature and diversity of the intestinal microbiota (125). After a severe transient gut infection, a different stable ecosystem can take over (dysbiosis), which can keep the host alive, but leads to illnesses such as irritable bowel syndrome and IBD (126), with consequent susceptibility to inflammation-related cancer. Hygiene hypothesis, depression, and cancer Depression provides another important potential link between the Hygiene hypothesis and cancer. Depression predicts increased mortality in cancer patients. This is true whether depression is diagnosed before or after cancer diagnosis and remains after controlling for confounding medical variables (127). An analysis of 25 independent studies reported that mortality rates were up to 25% higher in patients experiencing depressive symptoms (P < 0.001) and up to 39% higher in patients diagnosed with major or minor depression 154 (P = 0.03) (128). Chronically raised levels of circulating proinflammatory cytokines can lead to depressive symptoms, and there is a subset of individuals with depression in whom the mood disorder may be caused by the same failure to terminate inappropriate inflammatory responses that lies behind the increases in somatic inflammatory disorders (allergies, IBD, autoimmunity) frequently discussed in relation to the Hygiene hypothesis (129, 130). Several anti-inflammatory agents such as anti-TNF and NSAIDs have now been found to exert anti-depressant effects (131). Interestingly, in an animal model heat-killed Mycobacterium vaccae, a potent inducer of Tregs (93), had anti-depressant effects similar to those of Prozac, and a subset of serotonergic neurons involved in this effect was identified (132). We can therefore postulate that by inducing regulatory mechanisms and reducing circulating pro-inflammatory cytokines, the microbial exposures involved in the Hygiene hypothesis may exert a supplementary anti-cancer effect, secondary to improvements in mood and coping. Microorganisms and immunotherapy of cancer The arguments and data presented above suggest that microbes or their components could have a role to play in cancer prevention by exerting immunoregulatory effects that suppress chronic oncogenic inflammation. Similarly, these preparations might aid treatment of established cancer by altering the balance of effector mechanisms, suppressing those that are unhelpful or detrimental (Th2), and driving effective anti-tumor effector pathways. In principle it might be possible to drive subsets of Tregs that block inflammation in the tumor bed (and so limit vascularization, and the provision of growth factors), without blocking the generation of effector cells in the lymphoid tissue (84). This aspect has not yet been investigated. Many research groups are trying to use microorganisms or their components as adjuvants and as carriers of tumor target antigens. The organisms selected are often intracellular infections. Thus, Listeria monocytogenes is being developed as a carrier of tumor antigens that enters APCs (133, 134), and components of Toxoplasma gondii are being used in a dendritic cell-based therapeutic cancer vaccine (135). However, most studies on the use of microorganisms for immunotherapy in human have used mycobacteria. Cancer immunotherapy with mycobacteria The best-established use of immunotherapy is intravesical BCG for bladder cancer. The mode of action is unclear, but a 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 Rook & Dalgleish Æ Infection, immunoregulation, and cancer recent review of 80 randomized trials and 11 meta-analyses concluded that intravesical BCG, although more toxic, is more effective than mitomycin-C for the prophylaxis of tumor recurrence in high-risk patients, although there is no significant difference in overall survival. Chemotherapy and BCG seem to be equally effective in preventing recurrence in intermediate risk groups (136). BCG has been tested in many other cancer types and is still sporadically used in melanoma. An examination of all reported trials available found a trend to enhanced survival in patients with stage III malignant melanoma and cutaneous non-visceral metastases given intralesional BCG (137). More recently, cancer studies have been concentrating on a different mycobacterium (M. vaccae) that has unusual immunological properties. Used as a heat-killed preparation, it drives Tregs and inhibits Th2 (93) but encourages cytotoxic T cells (138) and NK activity (139). Encouraged by several studies of heat-killed M. vaccae in animal models of various cancers, by Phase II studies on intradermal M. vaccae in prostate cancer (140), and in inoperable non-small cell lung cancer and mesothelioma (141), an open label Phase III trial was performed in a total of 419 patients with several types of lung cancer, mostly squamous cell carcinoma and adenocarcinoma (142). Deficiencies in the study design led to poor compliance, with only 53% receiving more than two injections of M. vaccae, and the analysis plan did not allow for independent appraisal of the different cancer types. Overall there was no survival advantage in the group receiving M. vaccae, but patients showed significantly less deterioration in their Global Health Status score (P = 0.02), particularly those parameters related to mood, coping, and depression (see previous section). Meanwhile, a separate analysis of compliant patients confirmed that while treatment with M. vaccae gave no survival benefit in squamous cell carcinoma, in sharp contrast, survival of compliant adenocarcinoma patients receiving M. vaccae was increased by a mean of 135 days (P = 0.0009, Kaplan–Meier log rank test) (143). This analysis fell outside the original analysis plan of the Phase III trial and must therefore be treated with caution, but it has been sufficient to motivate further trials that are now being planned. Heat-killed M. vaccae was also tested as an immunotherapeutic agent for patients with metastatic, postnephrectomy, renal cell carcinoma. Sixty patients received injections of M. vaccae intradermally on entry and after 2, 4, 8, 12, 16, 20, and 24 weeks. Subsequently, they received injections every 8 weeks. Survival was compared with that of historical con- 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 trols who had been treated either with biological response modifiers (IL-2, IFN-a) or with chemotherapy. Those treated with M. vaccae alone survived as long as those receiving IL-2 or IFN-a, and both treatment groups survived longer than those on chemotherapy (P < 0.001) (144). In a second small study, 36 patients were randomized to receive treatment with IL-2 alone or IL-2 plus M. vaccae. Those receiving IL-2 alone had significantly more adverse events than those receiving M. vaccae plus IL-2 (P < 0.001) (144). This combination therapy was prompted by a previous pilot study suggesting that the combination of M. vaccae and IL-2 might be more beneficial than M. vaccae alone in melanoma (145). A small biotech startup (Immodulon; http://www.immodulon.com/index.php) is currently setting up trials of cancer immunotherapy using a very similar organism. Conclusions In this review, we have brought together information from several different disciplines. Until now, the thinking that has been applied to the increases in chronic inflammatory disorders in rich developed countries has been entirely different from the thinking applied to similar increases in many cancers, despite rather precise epidemiological parallels. Here we have discussed the ability of infections to cause cancer but also pointed out that the immunodysregulation that accompanies the Western lifestyle might also contribute both to oncogenesis and to subsequent inability to control or eliminate incipient cancers. A greater understanding of these interactions might pave the way to improved microbe-based immunotherapies. Interestingly, the intracellular infections are central to this analysis and particularly to therapeutic studies currently in progress, and these results may provide valuable new tools. An obvious limitation of this discussion is the fact that most of the evidence that Treg and anti-inflammatory cytokines can sometimes protect against development or progression of cancer comes from animal models. We cannot yet state with certainty that the same is true for any human cancers. Nevertheless, the situation is changing, and as attention is focused more precisely on the exact location and phenotype of the Treg in human disease, the heterogeneity is beginning to emerge, and evidence of protective roles in some cancers is becoming suggestive. 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