Infection, immunoregulation, and cancer

� 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011 141

Graham A. W. Rook

Angus DalgleishInfection, immunoregulation, andcancer

Authors’ addresses

Graham A. W. Rook1, Angus Dalgleish2

1Department of Infection, University College London (UCL),

London, UK.2St George’s Healthcare NHS Trust, London, UK.

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.

Immunological Reviews 2011

Vol. 240: 141–159

Printed in Singapore. All rights reserved

� 2011 John Wiley & Sons A/S

Immunological Reviews

0105-2896

Summary: As man has moved rapidly from the hunter–gatherer envi-ronment to the living conditions of the industrialized countries, the inci-dences of some cancers have increased alarmingly. Recent increases areusually attributed to dietary changes or to altered exposures to putativecarcinogens associated with the modern lifestyle. However, the changesin cancer incidence parallel similar increases in non-neoplastic chronicinflammatory disorders (inflammatory bowel disease, allergies, andautoimmunity), and the epidemiology is often strikingly similar. Thisparallel is worth exploring, because the increases in chronic inflamma-tory disorders are at least partly explained by immunoregulatory defectsresulting from diminished exposure to microorganisms that co-evolvedwith mammals and developed a role in driving immunoregulatory cir-cuits (the hygiene hypothesis). Dysregulated chronic inflammation candrive oncogenesis and also provides growth and angiogenic factors thatenhance the subsequent proliferation and spread of tumor cells. Thus, amodern failure to downregulate inappropriate inflammation couldunderlie increases in some cancers in parallel with the increases inchronic inflammatory disorders. This possibility is supported by recentwork showing that in some circumstances regulatory T cells protectagainst cancer, rather than aggravating it, as previously assumed. Agreater understanding of these interactions might pave the way toimproved microbe-based immunotherapies.

Keywords: cancer, immunoregulation, Treg, hygiene hypothesis, infection, IL-10,TGF-b

Introduction

As man has moved rapidly from the hunter–gatherer environ-

ment to the living conditions of the rich industrialized coun-

tries, the incidences of some cancers have increased

alarmingly. Some cancers (< 20%) are attributed to onco-

genic 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 puta-

tive carcinogens associated with the modern lifestyle. Interest-

ingly, the changes in cancer incidence have been paralleled by

even larger increases in a number of non-neoplastic chronic

inflammatory disorders (inflammatory bowel disease, aller-

gies, and autoimmunity), and the details of the epidemiology

are often strikingly similar. This parallel is worth exploring,

because chronic inflammation can contribute to oncogenesis

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 pres-

ent throughout human evolution as commensals (notably in

the intestinal microbiota), environmental ‘pseudocommen-

sals’, causative agents of subclinical infections or asymptom-

atic carrier states, and helminth infections. Modern revisions

of the ‘Hygiene’ or ‘Old Friends’ hypothesis suggest that

such organisms have been tasked by co-evolutionary pro-

cesses with establishing the ‘normal’ levels of immunoregu-

lation 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 can-

cer 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 trans-

formation 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 Organism Cancer References

Infect and transform lymphoid cells Epstein–Barr virus (EBV) Burkitt’s lymphoma* (4)Human Herpesvirus 8 (HHV8) Kaposi sarcomaHuman T-lymphotropic virus 1 (HTLV-1) T-cell leukemia

Transformation HPV Cervical cancer (2)Inflammation and partial integration HBV Hepatocellular carcinoma (2)Chronic inflammation and oncogenic proteins Hepatitis C virus (HCV) Hepatocellular carcinoma (4)Chronic stimulation of lymphocytes bypathogen antigens and ⁄ or autoantigens

Helicobacter pylori Gastric lymphoma (3)Campylobacter jejuni � Immunoproliferative small intestinal dis. (29)Borrelia burgdorferi Cutaneous lymphoma (3)Chlamydia psittaci Ocular lymphoma (30)Hepatitis C virus (HCV)� Spleen lymphoma (3)

Chronic inflammation Bacteroides fragilis (enterotoxigenic) Colorectal cancer (146, 147)Malaria (cofactor) Burkitt’s lymphoma (7)Tuberculosis Lung cancer (9, 10)Schistosomiasis Bladder cancer (2, 5)Liver flukes Cholangiocarcinoma (5)Salmonella spp. Gallbladder cancer (8)Helicobacter pylori Gastric and esophageal adenocarcinoma (113, 115)

Chronic inflammation; possible associations Propionibacterium acnes? Prostate (148)Mycoplasma spp. ? Prostate (149)?? unknown prostatitis ?? Prostate

Immunosuppression HIV EBV+ CNS lymphomas (4)HHV8+ sarcoma (Kaposi)HPV+ ano-genital cancers

*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.

Rook & Dalgleish Æ Infection, immunoregulation, and cancer

142 � 2011 John Wiley & Sons A/S • Immunological Reviews 240/2011

volume, though there are exceptions. Malaria can act as a co-

factor for EBV-induced oncogenesis, because Plasmodium falcipa-

rum 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 immuno-

regulatory 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 onco-

genesis (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 adenocarci-

noma 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 fluores-

cent in situ hybridization (FISH) with probes for H. pylori 16S

rRNA and cagA, H. pylori was detected inside metaplastic, dys-

plastic, 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 observa-

tion 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).

Tumor necrosis factor-a (TNF-a) may be particularly impor-

tant (14), and it has been found to be secreted by a wide vari-

ety 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-sup-

pressor genes such as Von Hippel-Lindau tumor suppressor

(VHL), transforming growth factor-b (TGF-b), and phospha-

tase 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 (VEG-

FR1)-positive hematopoietic progenitors and inflammatory

Fig. 1. Some potential roles of infection in preventing and causingcancer (excluding direct transformation, which is discussed briefly inthe main text). Epidemiological studies indicate that transient infectionmay provide some protection against cancer. However, chronic infectionleading to chronic inflammation can initiate oncogenesis, and also, bysubsequently providing growth and angiogenic factors, potentiate exist-ing cancers.



cells to sites of tumor growth. These cells may also secrete fac-

tors 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 hun-

dreds of target genes related to cell growth, apoptosis, inva-

sion, and inflammation. These processes accelerate the

intrinsic mutation rate in cancer cells, at least partly by

increasing levels of reactive oxygen species (ROS) and nitro-

gen 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 (IL-

6) 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 microsat-

ellite instability in tumor suppressor genes can lead to uncon-

trolled 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 hypermuta-

tion 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 instabil-

ity results in abnormal segregation of chromosomes and aneu-

ploidy (12).

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 vas-

cular 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 inappro-

priate 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 signifi-

cant decrease in the number of pulmonary metastases (26,

27). Angiogenesis is further promoted by infiltration of

monocytes expressing tyrosine kinase with immunoglobulin-

like and EGF-like domains 2 (TIE-2). These TIE-2-expressing

monocytes (TEMs) constitute a small pro-angiogenic mono-

cyte 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 mecha-

nisms 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 Helico-

bacter infection (28).

Chronic stimulation of lymphoid cells

Chronic stimulation by microbial antigens or autoantigens is

associated with a distinct subset of extranodal lymphomas



arising from B cells of the marginal zone of mucosa-associ-

ated 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 ongo-

ing 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 cross-

reactivity with gastric autoantigens (31, 32). Indeed autoim-

munity, even when not directly attributable to a known

infection (e.g. Sjogren’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, eradi-

cation 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 bacte-

rial 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–Barre syndrome, which is caused

by cross-reactive antibodies that bind both to C. jejuni lipo-

oligosaccharides 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 occa-

sionally 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

resembling various types of differentiated small intestinal epi-

thelial 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 archi-

tecture. At some point, adenocarcinoma develops in these

lesions. Some investigators believe that the initial stages of

inflammation and atrophy create an abnormal microenviron-

ment 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 sus-

ceptible to mutation and deviation away from the pathway of

complete differentiation, resulting in progressive loss of dif-

ferentiation, uncontrolled replication, and eventual neoplastic

invasive behavior (36). If this is correct, then infection-

induced chronic inflammation can cause cancer by attrition of

local stem cells, and their replacement with more dysplasia-

susceptible 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 hypothe-

sis. 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 can-

cer 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 peo-

ple 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



0.32 respectively, and the trend was statistically significant

(P = 0.004) (38).

LPS, like many microbial components, is a powerful adju-

vant. 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 protec-

tive 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 hematologi-

cal tumors, though the validity of the findings remains uncer-

tain (43). To date, one can only speculate as to what effect

latent tuberculosis, affecting one third of the world’s popula-

tion, has on the incidence of cancer.

Cross-reactivity

It appears that glycosylation changes are early consequences of

oncogenesis, and glycoproteins on tumor cells bear oligosac-

charides that are markedly different from those found on pro-

teins produced by normal cells (44). It is suggested that these

may sometimes resemble the oligosaccharide structures of

bacteria (45), with more exposed galactose, N-acetylgalactos-

amine, 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 HERV-

K-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 vac-

cine, 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 pro-

tected 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

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 simulta-

neous 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. Cro-

hn’s disease and ulcerative colitis) (49). All of these chronic

inflammatory disorders show evidence of defective immuno-

regulation. Regulatory defects have been found in individuals

suffering from allergic disorders (50), some autoimmune dis-

eases (51, 52), and probably in IBD too (53, 54). This sug-

gests that in developed countries there is a generalized defect

in regulation of the immune system that is manifested in dif-

ferent 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 popula-

tion 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 syn-

drome 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 mamma-

lian evolutionary history had to be tolerated and so co-evolved

a role in driving regulatory pathways within the immune sys-

tem. The depletion of these organisms from the modern envi-

ronment 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 aller-

gies (56–58), Type 1 diabetes (59), or IBD (60–62). Rather

epidemiological, experimental, and clinical studies have indi-

cated that the important organisms in this context are rela-

tively benign organisms such as lactobacilli, helminths,

orofecally transmitted infections inducing carrier states, and



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 epi-

demiological transition: during industrialization, the con-

struction of modern cities, concrete, tarmac roads, and

separation from mud animals and feces that had always previ-

ously provided contact with them (Fig. 3).

Pro-inflammatory effects of previously beneficial genetic

factors

The depletion of regulation-inducing organisms leads to a fur-

ther complication. In the developed countries, genetic factors

that were not previously causing disease can assume negative

roles when the Old Friends are removed. In areas with the

highest burdens of immunoregulation-inducing organisms,

accumulation of compensatory single nucleotide polymor-

phisms (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 inade-

quate immunoregulatory mechanisms, causing, in some indi-

viduals, 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 possibil-

ity that mechanisms described by the Old Friends Hypothesis

may be protective by terminating inappropriate inflammation,

Fig. 2. The Hygiene or Old Friends hypothesis. The organisms with which mammals co-evolved (see Fig. 3) had to be tolerated, and mammals arenow in state of evolved dependence on their ability to drive immunoregulatory pathways. In developed countries, immunoregulation is thereforedecreased, and gene–environment interactions cause a subset of people living in developed countries to develop chronic inflammatory disorders. Thenature 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 (150, 151)Helminths (152, 153)Helicobacter pylori (117)Salmonella (154)Gut microbiota (123, 155)Mycobacterium tuberculosis (156, 157)Toxoplasma gondii (57, 151, 158)(unknown ? microbial)* (48, 159)

*Protection from cowsheds and homes contaminated by animal feces.



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,

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).

Fig. 3. The Darwinian background to the Hygiene or Old Friends hypothesis. The organisms implicated by epidemiology, clinical studies, in vitroexperiments, and animal models are shown highlighted in bold font on the right. These organisms accompanied man at least as far back as the Paleo-lithic 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 inthe Neolithic are not so relevant. The important organisms were progressively lost from the start of the second epidemiological transition in the early19th century.

present from the paleolithic Genetics

Potential susceptibility to Th1/Th17 or Th2 disorders in the

Compensatory SNP or alleles 1) pro-inflammatory cytokines

2) IgE

Environmental triggers

on their role in induction of immunoregulation

progressively from mid 18th C as contact withsoil, animals and faeces diminished

Delayed exposure to viruses ?

Allergies, MS, IBD, type 1 diabetes etc and some cancers?

Diet, obesity

Factors not usually sufficient to cause disease

Vit D deficiency,

Pollutants (dioxins) etc

Fig. 4. The additional effects of compensatory single nucleotide polymorphisms (SNPs) and of aggravating environmental changes. In parts ofthe 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 thenbecome risk factors for chronic inflammatory disorders. This situation is likely to be aggravated by other modern environmental changes that also tendto increase inflammatory responses, such as obesity, vitamin D deficiency, and pollutants such as dioxins that drive Th17 cells.



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 acti-

vated macrophages and is attenuated by administering anti-

IFN-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. Interest-

ingly, 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-inflamma-

tory effect, IL-10 is also involved in other protective anti-

tumor mechanisms, and further studies do indeed confirm

this finding. First, there is strong evidence that IL-10 encour-

ages 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 administra-

tion 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 num-

bers 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 indi-

cator 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 func-

tions of NK cells by expressing membrane-bound TGF-b

(76). Moreover, TGF-bs are involved in epithelial to mesen-

chymal 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

transition in vitro in numerous types of cancer cells (77). Simi-

larly, 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 initia-

tion. 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). Inter-

estingly, 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-inflamma-

tory 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 can-

cers 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 particu-

larly 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



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. felis-

infected 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 atro-

phic lesions were not seen (81). The co-infection did not

eliminate inflammation. Indeed, 16 weeks after infection gas-

tric colonization with H. felis was greater in the group co-

infected with H. polygyrus, particularly in the antrum, with

extension into the corpus mucosa. However, the inflamma-

tion 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 bac-

terial pathogen of mice. 129 ⁄ SvEv RAG-2-deficient mice were

infected with H. hepaticus by gastric gavage. Many of these ani-

mals 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 mecha-

nism discussed above in relation to the Hygiene hypothesis)

or passive transfer of Tregs can convert an inflammatory

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. How-

ever, the number of epithelial adenomas is reduced when het-

erozygote 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 inflam-

matory 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 adenocar-

cinoma 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-inflamma-

tory T cells), only Tregs from Helicobacter-experienced mice

were protective (Fig. 6). When derived from H. hepaticus-

naive ‘hygienic’ animals, they exacerbated the cancer in the

first two models and had no effect in the third one. The

Treg from animals that have experienced infection

Beneficial roles of Treg Detrimental roles of Treg

Treg in tumour stroma or local lymphoid aggregates block development of protective cytotoxic lymphocyte (CTL) response.

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-ββ: Anti-inflammatory Inhibit epithelial growth

Treg from ‘hygienic’ animals

IL-10: Inhibit protective anti-tumour response

TGF-ββ: Inhibit CTL

Fig. 5. The heterogeneous roles of Tregs and anti-inflammatory cytokines in cancer. It is well documented that Tregs can oppose protectiveresponses to cancer. It is now emerging that Tregs of certain poorly defined phenotypes, in certain locations, can be protective against both initiationand progression of cancer. Tregs can terminate chronic inflammation and so stop oncogenesis. In existing tumors, Tregs can block release of growthand angiogenic factors, but to achieve this, they need to be in the tumor bed itself. Tregs located in surrounding lymphoid aggregates are more likelyto 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 ani-mal models (see Fig. 6), Tregs from infected animals can have the relevant properties, whereas Treg from very clean specific-pathogen free (SPF) ani-mals do not.



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, indepen-

dent 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 can-

cer-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 associ-

ated with Foxp3+ cells in lymph nodes and in tissue surround-

ing the tumor, whereas remission was associated with Foxp3+

cells in the tumor itself (23). Female FVB strain MMTV-HER-

2 ⁄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. Interest-

ingly, 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 simi-

lar phenomenon in the APCMin ⁄ + mouse model (23).

This observation might be relevant to human disease. The

assessment of numbers of circulating Tregs or of Tregs infil-

trating 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, pan-

creas, stomach, liver, and ovary, whereas Tregs appeared ben-

eficial 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 het-

erogeneity of results suggests that something is being missed,

and the animal models discussed above suggest that the locali-

zation of the Tregs might be crucial. A study that counted

infiltrating Tregs in breast cancer concluded that high num-

bers of these cells were detrimental to survival (85), but a fur-

ther 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 inflamma-

tion discussed earlier.

The microanatomical location is also crucial when consider-

ing the role of tumor-infiltrating macrophages. These corre-

late 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 enhanc-

ing 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 adenoma-

tous polyposis, there is focal mastocytosis that promotes

tumor initiation and progression (87, 88). Adoptively trans-

ferred Tregs suppress this mastocytosis. In contrast, the

endogenous Tregs that are present in the polyps in large num-

bers show proinflammatory activity and promote differentia-

tion 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 inflamma-

tion, perhaps because TNF and IL-6 are being released sponta-

neously 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

Infect with Helicobacter hepaticus

Treg-deficient effector T cells (CD4+CD45RBhi)

C57Bl APCmin+/–

129 Rag2–/–

Colorectal cancer

Mammary adenocarcinoma

Transfer of Treg from infected mice is protective

Fig. 6. Examples of beneficial effects of Tregs in animal models ofcancer. Transfer of Tregs from animals infected with Helicobacter hepaticusinto animals developing the cancers was protective, even when this organ-ism was not involved in the cancer concerned and was not present in therecipient. By contrast, Tregs from specific pathogen-free animals were notprotective, indicating that the precise phenotype of the Treg is crucial.



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 influ-

ence the balance between different effector cell types and

selectively alter cytokine secretion profiles. For instance,

Foxp3+ Tregs can functionally differentiate to acquire an abil-

ity 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 infil-

trate 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 phe-

notype (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

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. Inci-

dences 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 Hispan-

ics, 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 resis-

tance 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 neo-

nates can result in serious disease (e.g. polio or hepatitis A

virus) and delayed exposure to viruses has also been consid-

ered 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 epidemiolog-

ical findings that gave rise to the original version of the

Hygiene hypothesis (101, 104). A study in Northern Califor-

nia 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



attendance appeared to give some protection against not only

ALL but also to a limited extent against other pediatric can-

cers such as central nervous system tumors (106, UKCCS

investigators, unpublished data quoted in 104). Equally strik-

ing is the fact that an analysis of data from 40 countries

revealed a very significant positive correlation between inci-

dences of ALL and Type 1 diabetes. Both diseases also corre-

lated 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 ende-

mic. 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 town-

ship 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 simulta-

neous helminth infections, as demonstrated in experimental

models outlined earlier (23, 81). The milder chronic inflam-

mation 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 asso-

ciated 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 dam-

age and also causing the infection to be more frequently con-

fined 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 inci-

dence 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 esopha-

gitis and carcinogenesis (Fig. 7).

Loss of helminths ------------> --- Loss of H. pylori (antibiotics) --->

Normal (ancient) Late 20th C

Gastric atrophy - B12 deficiency - Iron deficiency - Some adeno-

carcinoma in old age

Peptic ulcer disease shows sharp peak

Sustained high acid, oesophageal adenocarcinoma

Progressive loss of Treg, more allergic disorders

Mid 19th–early 20th C

Fig. 7. The changing role of Helicobacter pylori. Human studies and animal models suggest that in the presence of co-infection with Treg-inducinghelminths, the inflammatory response induced by H. pylori is attenuated but involves the whole stomach. Adenocarcinoma is a late consequence insome individuals. As helminths are progressively eliminated, a more vigorous response occurs, and peptic ulceration is seen in individuals whose Tregresponse to H. pylori itself is suboptimal, while H. pylori becomes confined to the pylorus. More recently, the situation has been complicated by the factthat long-term infection with H. pylori results in decreased acid production. Thus, total loss of H. pylori due to antibiotics results in sustained high levelsof acid production and consequent esophageal adenocarcinoma (modified and redrawn from reference 113).



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, epide-

miological 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 pro-

vide 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 coun-

tries undergo the second epidemiological transition to eco-

nomic 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 immunoreg-

ulation in a mouse model (81), and helminths have a thera-

peutic effect in human IBD (121, 122). However, other

components of the microbiota also have important immuno-

regulatory 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 (dysbio-

sis), 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 experienc-

ing depressive symptoms (P < 0.001) and up to 39% higher

in patients diagnosed with major or minor depression

(P = 0.03) (128). Chronically raised levels of circulating pro-

inflammatory 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 investi-

gated.

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 infec-

tions. Thus, Listeria monocytogenes is being developed as a carrier

of tumor antigens that enters APCs (133, 134), and compo-

nents 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



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 signifi-

cant difference in overall survival. Chemotherapy and BCG

seem to be equally effective in preventing recurrence in inter-

mediate 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 mela-

noma and cutaneous non-visceral metastases given intrale-

sional BCG (137).

More recently, cancer studies have been concentrating on a

different mycobacterium (M. vaccae) that has unusual immu-

nological 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 pros-

tate cancer (140), and in inoperable non-small cell lung can-

cer 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 adeno-

carcinoma (142). Deficiencies in the study design led to poor

compliance, with only 53% receiving more than two injec-

tions 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 analy-

sis 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 immunotherapeu-

tic 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-

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 sig-

nificantly 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 combi-

nation 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 disor-

ders in rich developed countries has been entirely different

from the thinking applied to similar increases in many can-

cers, despite rather precise epidemiological parallels. Here we

have discussed the ability of infections to cause cancer but

also pointed out that the immunodysregulation that accom-

panies the Western lifestyle might also contribute both to

oncogenesis and to subsequent inability to control or elimi-

nate incipient cancers. A greater understanding of these inter-

actions 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. Never-

theless, 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. We hope that this review will encour-

age further explorations of this area which has the potential to

influence both the prevention and treatment of those cancers

for which it proves relevant.



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Infection, immunoregulation, and cancer

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