(PDF) Autoimmune Disease and the Human Metagenome

Life Sciences - Ecology | Metagenomics of the Human Body

approx.

Nels on, Ka ren E. ( Ed. )

1st Edition., 2011, 350 p. 40 illus., 20 in color., Hardcover

ISBN: 978-1-4419-7088-6

Not yet publish ed. Available: D ece mber 26 , 20 10

$209.00

Metagenomics of the Human Body introduces readers to the major findings from the human genome project and at the same time

presents the crossover to the human metagenome/microbiome, which we are only starting to understand through the advent of

newly emerging technologies and other developments. The book brings a new perspective by combining the information gained

from the human genome with that derived from parallel metagenomic studies, and new results from investigating the effects of

these microbes on the host immune system. As the field of metagenomics continues to evolve, Metagenomics of the Human Body

brings together leaders in the field and their unique perspectives on this topic. The authors focus on the human genome and

recent developments in the fields of microbial ecology and metagenomics of the microbial species that are associated with the

human body. They also discuss the enormous implications for health and disease. Metagenomics of the Human Body is ideal for

scientists, clinicians, community activists, undergraduate and graduate level students, as well as ethical and legal groups

associated with or interested in the issues surrounding the human genome. About the Editor Dr. Karen E. Nelson is the Director of

the Rockville Campus of the J. Craig Venter Institute (JCVI) where she has been for the past 14 years. She was formerly the

Director of Human Microbiology and Metagenomics in the Department of Human Genomic Medicine at the JCVI. She has authored

or co-authored over 100 publications, and is currently Editor-in-Chief of the Springer journal Microbial Ecology. She is also a

standing member of the NRC Committee on Biodefense, a member of the American Society for Microbiology (ASM)

Communications Committee and a Fellow of the ASM.

Content Level » Professional/practitioner

Related subjects » Ecology

TABLE OF CONTENTS

Preface: The Human Genome and the Human Microbiome.- Chapter 1: The Human Genome, Microbiome and Disease.- Chapter

2: Host Genotype and the effect on Microbial Communities.- Chapter 3: The Human Microbiome and Host-Pathogen Interactions.-

Chapter 4: The Human Virome.- Chapter 5: Selection and Sequencing of Strains as References for Human Microbiome studies.-

Chapter 6: The Human Vaginal Microbiome.- Chapter 7: The Human Lung Microbiome.- Chapter 8: The Human Skin Microbiome

in Health and Skin Diseases.- Chapter 9: The Human Oral metagenome.- Chapter 10: Infectogenomics: aspect of Host Responses

to Microbes in the Digestive Tract.- Chapter 11: Autoimmune Disease and the Human Metagenome.- Chapter 12: Metagenomic

applications and the potential for understanding chronic liver disease.- Chapter 13: Symbiotic gut microbiota and the modulation of

human metabolic phenotypes.- Chapter 14: MetaHIT: The European Union Project on Metagenomics of the Human Intestinal

Tract.- Chapter 15: Implications of Human Microbiome Research for the Developing World.

About this book

http://www.springer.com/life+sciences/ecology/book/978-1-4419-7088-6

1 of 1 8/17/2010 7:55 A

M

Autoimmune disease and the

human metagenome

Amy D. Proal, Murdoch University

Paul J. Albert, Weill Cornell Medical College

Trevor G. Marshall, Murdoch University

...........................................................................................................................................................................................Abstract 2

...................................................................................................................................................................................Background 2

.............................................................................................Culture-independent methods for identifying microbes 4

........................................................................................................................................................The human metagenome 6

.................................................................................................................................................................Microbial complexity 7

...........................................................................................Towards a more nuanced view of the human microbiota 8

...............................................................................Pathogens alter the expression of human genes and receptors 9

...............................................................Successive infection and variability in disease onset and presentation 13

..................................................................................Early infections predispose a person to later chronic disease 14

................................................................................................................................................................................Comorbidity 15

................................................................................................................................................Causation versus association 17

......................................................................................................................................Microbial interaction and disease 17

................................................................................................................................................................Familial aggregation 18

.....................................................................................................Is autoimmune disease predisposition Mendelian? 19

...........................................................................................................................................SNPs and autoimmune disease 19

.................................................................Potential systematic errors in the interpretation of the metagenome 20

.......................................................................................................................Antibodies in response to microbial DNA 21

........................................................................................................................Therapies in the era of the metagenome 23

....................................................................L-form bacteria: an often overlooked component of the microbiota 25

.................................................................................A research consideration: men are not tall mice without tails 25

....................................................................................................................................................................................Discussion 26

.................................................................................................................................................................Acknowledgements 27

.................................................................................................................................................................................Works Cited 27

This is a preprint of a book chapter published by Springer Publications, (c) Copyright, 2010, Springer Publications

Abstract

The prevailing theory of autoimmune disease, that the

body creates autoantibodies that attack “self,” was devel-

oped during an era when culture-based methods vastly

underestimated the number of microbes capable of per-

sisting in and on Homo sapiens. Thanks to the advent of

culture-independent tools, the human body is now

known to harbor billions of microbes whose collective

genomes work in concert with the human genome. Thus,

the human genome can no longer be studied in isolation.

Some of these microbes persist by slowing the activity of

the VDR nuclear receptor, aﬀecting the expression of en-

dogenous antimicrobials and other key components of

the innate immune system. It seems that bacteria that

cause autoimmune disease accumulate over a lifetime,

with individuals picking up pathogens with greater ease

over time, as the immune response becomes increasingly

compromised. Any one autoimmune disease is likely due

to many diﬀerent microbes within the metagenomic mi-

crobiota. This helps explain the high levels of comorbidity

observed amongst patients with autoimmune conditions.

What are commonly believed to be autoantibodies may

instead be created in response to this metagenomic mi-

crobiota, when the adaptive immune system is forced to

deal with disintegration of infected cells. Similarly, haplo-

types associated with autoimmune conditions vary widely

amongst individuals and populations. They are more sug-

gestive of a regional infectious model rather than a model

in which an illness is caused by inherited variation of HLA

haplotypes.

Background

In 1922, Ernst Almquist - a colleague of Louis Pasteur -

commented, “Nobody can pretend to know the complete

life cycle and all the varieties of even a single bacterial

species. It would be an assumption to think so.”1

While

Almquist's work on idiopathic bacteria in chronic disease

never received the plaudits accorded Pasteur's work, Alm-

quist foresaw the complexity that would later be inherent

to the ﬁeld of metagenomics - a ﬁeld that today forces us

to examine how countless microbial genomes interact

with the human genome across disease states.

Yet in the decades before novel genomic technology

made a metagenomic understanding of disease possible,

bacteria could only be cultured in vitro on a limited range

of growth media. As most major diseases of the time - tu-

berculosis, pneumonia, leprosy, and others - were linked

to the presence of a handful of acute pathogens able to

grow under these constraints, a "game over" attitude to-

ward infectious agents dominated the thinking of much

of the medical community. Little consideration was given

to the possible role of these pathogens in autoimmune

and inﬂammatory disease states. Instead, for most of the

twentieth century, the predominant feeling about the

treatment, control and prevention of diseases with a pos-

sible infectious etiology was optimism.2

Between 1940 and 1960, the development and successes

of antibiotics and immunizations added to this optimism

and, in 1969, Surgeon General William H. Stewart told the

United States Congress that it was time to “close the book

on infectious diseases.”3 With “victory” declared, increasing

emphasis was directed at the “non-infectious” diseases

such as cancer and heart disease. In many cases, research

on infectious disease or activities on their prevention and

control were de-emphasized and resources were reduced

or eliminated. As recently as the 1980s, pharmaceutical

companies, believing that there were already enough an-

tibiotics, began reducing the development of new drugs

or redirecting it away from antibiotics.

Despite this rosy narrative, some microbiologists were

never convinced that drugs like penicillin had ended the

war between man and microbe. In 1932, Razumov noted a

large discrepancy between the viable plate count and to-

tal direct microscopic count of bacteria taken from

aquatic habitats.4 He found higher numbers (by several

orders of magnitude) by direct microscopic counting than

by the plating procedure. In 1949, Winogradsky conﬁrmed

Razumov's assessment and noted that many microbes are

not satisﬁed with laboratory cultivation conditions. He

remarked that readily cultivated bacteria in natural micro-

bial communities “draw importance to themselves,

whereas the other forms, being less docile, or even resis-

tant, escape attention.”5

In 1985, Staley and Konopka

pointed to Razumov's discrepancy and called it the “Great

Plate Count Anomaly.”6

Their review describes work in

which they compared the eﬃcacy of a ﬂuorescent dye

versus standard plating procedures in detecting bacterial

species in samples of water collected from Lake Washing-

ton. They found that only approximately 0.1-1.0% of the

Proal et al.: Autoimmune disease and the human metagenome 2

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

total bacteria present in any given sample could be enu-

merated by the plating procedure - causing them to con-

clude that, unless new methods for detecting bacteria

were employed, "No breakthrough in determining species

diversity seems likely in the near future."

Meanwhile, some microbiologists continued their best

eﬀorts to alter the pH and growth medium of their sam-

ples in an eﬀort to look for previously undetected bacteria

in chronic disease states. Over the course of a career

spanning almost 50 years, Lida Mattman of Wayne State

University cultured wall-less forms of bacteria from the

blood samples of patients with over twenty inﬂammatory

diagnoses including multiple sclerosis and sarcoidosis.7

She authored an entire textbook on novel approaches for

in vitro cultivation of bacteria.1 Over his thirty-nine year

career at Tulane University, Gerald Domingue published

dozens of papers and book chapters devoted to the role

of chronic forms of bacteria in inﬂammatory disease. "It is

unwise to dismiss the pathogenic capacities of any mi-

crobe in a patient with a mysterious disease,” he wrote.

“Clearly, any patient with a history of recurrent infection

and persistent disability is sending the signal that the

phenomenon [infection with chronic bacteria or viruses]

could be occurring. The so-called autoimmune diseases in

which no organism can be identiﬁed by routine testing

techniques are particularly suspect.”8

Yet, scientists like Mattman and Domingue faced serious

challenges in trying to convince the medical community

their work was valid. Other research teams using less rig-

orous techniques often failed to duplicate their ﬁndings.

Many of their observations were dismissed on the premise

that their samples could have been contaminated. How-

ever, the greatest impediment towards the acceptance of

this work was a set of rules set in motion by 19th century

German physician Robert Koch. These rules, known as

"Koch's Postulates," stipulate that in order for a microbe to

be deemed a causative agent of a disease, certain criteria

must be met. The same microbe must be identiﬁed in

every person with a given disease; the speciﬁc microbe

must be able to be grown on pure culture medium in the

lab; and, when reintroduced into a healthy animal or per-

son, must produce the disease again.

While Koch's Postulates may have oﬀered a certain clarity

during the formative stages of the ﬁeld of microbiology,

the rules distracted scientists from considering the possi-

bility that multiple species could be responsible for the

onset of a single disease state. Even today, Koch's notions

about disease are regularly invoked9 despite the emer-

gence of a number of counterexamples. Neither Mycobac-

terium leprae, which is implicated in leprosy, nor Trepo-

nema pallidum, which causes syphilis, fulﬁll Koch's Postu-

lates, because these microbes cannot be grown in con-

ventional culture media. Viruses further invalidate Koch’s

postulates because most require another living cell in or-

der to replicate.10

In the absence of clear connections between a single mi-

crobe and a single disease, most microbiologists necessar-

ily assumed that the body was a sterile compartment and

that inﬂammation, which might well suggest the presence

of microbes, was attributed to an idiopathic causation.

Unable to grow all but a fraction of bacteria found in the

human body in the conﬁnes of a Petri dish, and con-

strained by a lack of technology with which to detect new

microbes, the theory of autoimmune disease, in which the

immune system loses tolerance and generates antibodies

that target self gained momentum in the 1960s.

Yet over the past decade, the role of infectious agents in

autoimmune disease has once again gained momentum.

The 2004 International Congress on Autoimmunity in Bu-

dapest was themed “Autoimmunity and Infection” with

many subsequent conferences and papers in the same

vein. However, nearly all speakers discussed the role of

viruses in autoimmune disease, while only a few contem-

plated bacteria. Autoimmune conditions were repeatedly

attributed to easily cultured viruses such as Epstein-Barr

and Herpes 6. Where bacteria were discussed, most re-

ports centered on select pathogens such as Chlamydia

pneumoniae. Yet because none of these pathogens have

ever been detected in any one autoimmune disease state

100% of the time, such researchers continue to paint

autoimmune diseases as a mosaic - in which the hallmarks

of infection are continually present in bits and pieces but

cannot be drawn into a fully cohesive picture. Yet the

emerging science of metagenomics is beginning to un-

mask entirely new populations of microbes whose ge-

nomes allow for a means by which to bridge these gaps.

The following chapter examines how this metagenomic

microbiota can cause the dysfunction seen in a wide

range of autoimmune conditions.

Proal et al.: Autoimmune disease and the human metagenome 3

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

Culture-independent methods for identifying

microbes

In 2007, a study orchestrated by NASA announced that

the surfaces of the supposedly sterile "clean rooms," in

which technicians assemble spacecraft, host an abun-

dance of hardy bacteria.11 Samples taken from clean

rooms at the Jet Propulsion Laboratory in California, the

Kennedy Space Flight Center in Florida, and the Johnson

Space Center in Houston revealed the presence of almost

100 types of bacteria representing all the major bacterial

phyla; 45 percent of the species identiﬁed were previously

unknown to science. The ﬁndings came as a shock to

NASA oﬃcials, who were left to wonder exactly how many

unknown microbes might have been taken to the moon

and Mars.

These clean room bacteria had not been previously de-

tected because they could not be characterized by stan-

dard cultivation techniques. To ﬁnd them, the research

team had used a genomic approach - RNA gene sequence

analysis - to characterize the genetic material of the bac-

terial species in the rooms previously touted as sterile.

Similar culture-independent tools are beginning to revo-

lutionize our understanding of autoimmune disease by

allowing for a vastly more comprehensive understanding

of the microbes that persist in Homo sapiens, microbes

that may cause the generation of autoantibodies. Ge-

nomic sequencing techniques, including 16S RNA se-

quencing, polymerase chain reaction and, more recently,

pyrosequencing, have made it clear that only a fraction of

those microbes that persist in the human body will grow

on the limited medium of a Petri dish. With the advent of

these technologies, the ﬁeld of metagenomics was born.

Rather than focusing on the study of single microbes and

their genomes, metagenomics provides a means of ana-

lyzing aspects of microbial communities through their

underpinning genetics. The amount of novel microbial

genetic information that is generated on a daily basis by

metagenomic analysis is so great that multidisciplinary

approaches that integrate statistics, bioinformatics, and

mathematical methods are required to assess it eﬀec-

tively.

Today, the National Institutes of Health (NIH) estimates

that a mere 10% of the cells that comprise Homo sapiens

are human cells. The remaining 90% are bacterial in origin.

The number of E. coli in a single human is comparable to

the entire human global population – approximately six

billion people.12

Such knowledge has forever changed the

manner in which the human organism is perceived. We

may best describe the human being as a super-organism

in which communities of diﬀerent organisms ﬂourish in

symbiosis with the host. Yet even with the availability of

technology to explore the microbial world in depth, to

date, only a fraction of the human bacterial microbiota

has been genetically identiﬁed and characterized. As of

late 2009, approximately 1,100 published complete bacte-

rial genomes had been identiﬁed with 6,000 more under

review.13 Nevertheless there are still huge gaps in our un-

derstanding of how the microbiota contributes to human

health and disease.

Viruses (the virome) and phages are also key components

of the microbiota. Like bacteria, many of these microbes

have yet to be fully characterized by high-throughput ge-

nome sequencing. However, molecular analysis has re-

vealed that nearly all humans acquire multiple viruses,

usually within the ﬁrst years of life, viruses that generally

remain with them throughout life. Polyomaviruses infect

between 72% and 98% of humans, surviving in the kid-

ney, lung, and skin.14 Similarly, human herpes viruses are

extremely persistent. Anelioviruses, as well as adeno-

associated virus are now recognized to infect most hu-

mans by the end of childhood. The role of these viruses is

unknown, but a signiﬁcant number of people who harbor

them become symptomatic later in life, suggesting that

they may be capable of virulence under conditions of

immune dysfunction. According to Herbert Virgin of

Washington University, "We carry, for good or for ill, many

lifelong [viral] passengers."14

In the next ﬁve years, researchers associated with the NIH

Human Microbiome Project (HMP), plan to use molecular

genetic sequencing in an eﬀort to catalog the bacterial

component of the human microbiome. This initiative

promises to increase our knowledge of bacterial diversity.

The NIH has funded many more HMP projects, with the

goal that the diagnosis, treatment and prevention of

many inﬂammatory diagnoses can be improved by exam-

ining how the microbiota diﬀers between those people

with a disease and their healthy counterparts. Thus far,

targeted conditions include Crohn's disease, inﬂammatory

Proal et al.: Autoimmune disease and the human metagenome 4

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

bowel disease, vaginosis, psoriasis, and other conditions

now considered to be autoimmune. Early work has al-

ready demonstrated fundamental discrepancies in micro-

bial composition between health and disease. Swidsinksi

et al. found that patients with irritable bowel syndrome

have more bacteria from diverse genera attached to their

epithelial gut surfaces than do healthy controls. Some of

these microbes, such as Bacteriodes, were found to pene-

trate the epithelial layer, at times intracellularly.15 Enck et

al. found that irritable bowel syndrome manifests with a

relative decrease in populations of biﬁdobacteria and sig-

niﬁcant diﬀerences in a variety of other microbes, includ-

ing those that cause the production of gas.16

Medicine has become comfortable acknowledging that

bacterial populations exist in the areas of the body in con-

tact with the external environment, such as the airways,

gastrointestinal tract, mouth, skin, and vagina/penis. For

example, analysis of the human oral cavity by Nasidze et

al. identiﬁed 101 bacterial genera in the mouth as well as

an additional 64 genera previously unknown to science.17

Yet microbes have also been shown to persist in many

other body tissues including joints and blood vessels.

Some of the same bacteria identiﬁed in the salivary mi-

crobiome, such as Actinobacillus actinomycetemcomitans

and Porphyromonas gingivalis - both of which cause tooth

decay18 - have also been identiﬁed in atherosclerotic

plaque.19 Bacterial DNA has been detected in the blood.20

Recently, 18 diﬀerent bacterial taxa were detected in the

amniotic ﬂuid, which was previously believed to be com-

pletely sterile.21 Analysis using 16S rRNA sequencing de-

tected 28 distinct phylotypes on bioﬁlm removed from

prosthetic hip joints during revision arthroplasties - joints

also removed from a body compartment also thought to

be sterile. The prevalence of hydrothermal vent eubacteria

- which were previously thought to persist only in the

depths of the ocean since they were found at tempera-

tures well above 176°F (80°C) - was higher than the preva-

lence of Staphylococcus aureus, a common bioﬁlm species

(Figure 1).

It is now more prudent to assume that tissues that be-

come inﬂamed in disease most probably do so because of

the actions of microscopic pathogens, rather than idio-

pathic causation. Diﬀerent microbial populations have

been identiﬁed in many non-gastrointestinal autoimmune

Proal et al.: Autoimmune disease and the human metagenome 5

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

Figure 1. Bacterial species identiﬁed by 16S rRNA gene sequencing of clones from 10 prosthetic hip joints191

conditions including sarcoidosis,22 ankylosing spondyli-

tis,23

and chronic fatigue syndrome, 24

rheumatoid arthri-

tis, multiple sclerosis, Hashimoto's Thyroiditis, and oth-

ers25. These diseases share features of microbial infection

including widespread inﬂammation and periods of re-

lapse. Sarcoidosis and Crohn's disease are characterized

by granuloma. In more than a dozen infectious diseases,

granuloma is widely acknowledged to be a host-

protective structure and to occur when acute inﬂamma-

tory processes cannot destroy invading agents.26

The human metagenome

At only approximately 23,000 genes, the human genome

is dwarfed by the thousands of genomes of the bacteria,

viruses, and phages that persist in and on humans. Given

the sheer number of microbial genes, it is no longer pos-

sible to study the human genome in isolation. Rather, the

human genome is only one of myriad genomes that inﬂu-

ence the Homo sapiens experience. Humans are controlled

by a metagenome - a tremendous number of diﬀerent

genomes working in tandem. Because they are so small,

thousands of microbial cells can persist inside a single in-

fected human cell.27 The combined genetic contributions

of these microbes invariably provide a vast number of

gene products not encoded by our own relatively small

genomes.

There is considerable similarity between the functions of

the bacterial organisms and the human organisms. For

example, humans and E. coli metabolize glucose-6-

phosphate in similar fashion, producing almost identical

metabolites.28

Thus, the transgenomic interaction be-

tween an E. coli genome and the human genome, as they

exchange nutrients and toxins, increases the complexi-

ty of transcription and translation for both species. The

dihydrofolate reductase (DHFR) antagonist trimethoprim

is such an eﬀective antibiotic because, like humans, bacte-

rial species possess a folate metabolism. Bacteria in the

distal intestinal tract of mice have been shown to signiﬁ-

cantly alter the composition of human blood metabolites

- including amino acids, IPA, and organic acids containing

phenol groups - providing another example of the signiﬁ-

cant interplay between bacterial and human metabolism.

A broad, drug-like phase II metabolic response of the host

to metabolites generated by the gut microbiota was ob-

served,29 suggesting that the gut microbiome has a direct

impact on the host's capacity for drug metabolism.

In the pre-genomic era, diseases were classiﬁed largely on

the basis of symptom presentation, while in recent dec-

ades, researchers have attempted to categorize them

based on common genes. Yet metagenomics dictates that

we must also consider how the many microbial metabo-

lites aﬀect expression of these genes. Some genes and

their related pathways have already been shown to inﬂu-

ence the pathogenesis of autoimmune disease. For exam-

ple, Goh et al. has shown that PTPN22 is related to rheu-

matoid arthritis, lupus, and diabetes mellitus.30

Yet expres-

sion of PTPN22 is also modiﬁed by the bacterial metage-

nome - it has been shown to be upregulated as part of the

innate immune response to mycobacteria.31 The impor-

tance of understanding how microbes aﬀect PTPN22

across multiple disease states has special impetus given

the increased rate of latent tuberculosis in the global

population as well as studies showing high rates of infec-

tion by Mycobacterium avium among autoimmune

patients.32

Many of the most well studied persistent pathogenic bac-

teria have evolved mechanisms to evade the immune re-

sponse and survive inside macrophages and other

phagocytic cells. These include Francisella tularensis,33 My-

cobacterium tuberculosis,8

Rickettsia massiliae,9

Brucella

spp.,34 Listeria monocytogenes,35

Salmonella typhimurium36

among others. This suggests that other disease-causing

components of the microbiota may also persist in the cy-

toplasm of nucleated cells, where they have access to

both human DNA transcription and protein translation

machinery.37 When Shigella persists within a macrophage

it modulates numerous host signaling pathways, includ-

ing those that inactivate mitogen-activated protein

kinases.38

Brucella spp. downregulates genes involved in

cell growth and metabolism, but upregulates those asso-

ciated with the inﬂammatory response and the comple-

ment system upon infecting a macrophage.

Additionally, there appears to be an entire intra-

cytoplasmic microbiota within phagocytic cells. Wirostko's

team at Columbia University in the 1980s and 1990s used

electron microscopy to identify entities within the cyto-

plasm of phagocytes from patients with juvenile rheuma-

toid arthritis, sarcoidosis,39

Crohn's, and other inﬂamma-

Proal et al.: Autoimmune disease and the human metagenome 6

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

tory diseases40. The wide variety of elongated and globu-

lar formations, together with both the existence and ab-

sence of exoskeletons around the microbiota, would im-

ply that the observed communities are metagenomic,

rather than due to any one single obligate phagocytic

pathogen.

Microbial complexity

The HIV genome consists of a single strand of RNA com-

prising 9 genes, from which are transcribed 19 proteins.

Transcription is non-contiguous, and variations abound.

For example, “Tat” is transcribed in multiple pieces that are

subsequently joined. Yet 1,443 direct interactions (3,300

total interactions) have been identiﬁed between just

these 19 proteins and the human metabolome.41 Consider

that the average bacterial genome codes for hundreds or

sometimes thousands of proteins. According to one re-

cent estimate, the average human gut microbiota codes

for 9 million unique genes.42 Factor in the proteins created

by viruses and phages, and eﬀorts to understand how

these proteins aﬀect the metabolome leave an observer

with little more than stochastic noise, particularly since

biological systems are replete with components showing

nonlinear dynamic behavior.

Subsequently, interaction between the metagenome and

the human genome introduces a new level of complexity

to the study of autoimmune disease - complexity that

renders it nearly impossible to fully comprehend the vast

number of the interactions between the human genome

and those microbial genomes capable of inﬂuencing the

pathogenesis of autoimmune disease. According to

Bunge, the size of a gene pool for a given environmental

sample can be estimated by mathematical modeling, but

the size of the gene pool for a microbial biosphere such as

the human body, may be beyond any current credible

model.43

While this complexity poses a signiﬁcant chal-

lenge to systems biology and to Koch's simplistic one

gene-one disease model, it does not impede the emer-

gence of a better understanding of the human superor-

ganism and the processes that cause disease.

Lifelong symbiosis between the human genome and per-

sistent components of the metagenome has shifted the

focus of microbiology away from the search for a single

pathogen in a disease state. Many research teams are now

striving to understand how components of the microbiota

may cause disease. For example, researchers with the

European Tract Meta Initiative are studying how bacteria

in the gut may contribute to obesity and inﬂammatory

bowel disease. The goal of the project is simply to exam-

ine associations between bacterial genes and human

phenotypes. “We don’t care if the name of the bacteria is

Enterobacter or Salmonella. We want to know if there is an

enzyme producing carbohydrates, an enzyme producing

gas or an enzyme degrading proteins,” explains Francisco

Guarner of the project.

Studies focused on enzymes, proteins and carbohydrates

are studies of the metabolome. Metabolomic approaches

can be used to characterize entire components of the mi-

crobiome that cannot easily be seen or studied directly.

Because the downstream results of gene expression mani-

fest in the human metabolome, the metabolome can be

analyzed for the presence of those unique metabolites

created under the inﬂuence of the microbiota. Dumas et

al. used mass spectroscopy to identify the non-human

metabolites present in the urine of subjects living in three

distinct populations - the United States, China, and

Japan.44 He found that subjects in each population pro-

duced very diﬀerent non-human metabolites. Thus, ge-

netic makeup, healthcare, nutrition, external toxins - fac-

tors associated with the acquisition of a particular micro-

biota - caused the three populations to become signiﬁ-

cantly diﬀerent. Moreover, when ﬁve of the Japanese sub-

jects moved to the United States, their metabolomes

changed to resemble those of the American population.

This suggests that the metagenome is indeed the product

of its environment, and that the composition of the mi-

crobiota is far more important than regional variations in

the human genome itself.

In another study, the INTERMAP epidemiological

study used a 1H NMR-based metabonomics approach to

examine diﬀerences in the urine metabolite proﬁles for

each of 4,630 participants from 17 populations in the USA,

UK, Japan and China.45 Elevated blood pressure was asso-

ciated with high levels of the bacterial co-metabolite for-

mate. Interestingly, low levels of hippurate and alanine,

which reﬂected gut microbial activities, were also found in

subjects with high blood pressure.46 This suggests that

certain microbial metabolites may serve as useful bio-

markers for a disease state.

Proal et al.: Autoimmune disease and the human metagenome 7

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

The fact that components of the microbiota are seldom

found as single entities further complicates the complex-

ity of transgenomic control in Homo sapiens. While just a

few decades ago, most of the bacteria in Homo sapiens

were assumed to persist on their own in a planktonic

form, it is now understood that large components of the

microbiota persist in communities commonly

called bioﬁlms -- they are sheltered by a self-created

polymeric matrix that better protects them from the im-

mune response. Hundreds of diﬀerent microbes can per-

sist in a single bioﬁlm community and individual bacteria

often form a niche inside the bioﬁlm that allows them to

promote their own survival and the chronic nature of the

infection. For example, more virulent bacteria may protect

the bioﬁlm from outside intrusion while other less in-

nocuous species inside the bioﬁlm may focus on obtain-

ing nutrients for the community. As the bioﬁlm forms and

then develops, the collective genetic expression of mi-

crobes in the bioﬁlm is altered dramatically. For example,

the expression of 800 genes have been shown to be al-

tered when a single bacterial species joins a

bioﬁlm.47 Bioﬁlms are increasingly being detected in auto-

immune diseases where they were not known to previ-

ously exist. For example, Wolcott recently used pyrose-

quencing to demonstrate that the infectious agents that

drive the development of diabetic leg, foot, and pressure

ulcers are almost all in a bioﬁlm state.48

Bacteria in bioﬁlm, their planktonic counterparts, viruses,

and other microbes rapidly and frequently share their

DNA with other species – even distantly related species –

through horizontal gene transfer. Genomic coherence is

further muddled by homologous recombination. This fur-

ther diversiﬁes the variability present in the human mi-

crobiome. Horizontal gene transfer is now believed by

many to occur so frequently that it has been proposed as

a means by which species can acquire new genetic traits.

Some argue that the number of microbes created through

homologous recombination is so high that the concept of

distinct bacterial species may become obsolete.49

Thus, the concept that a single pathogen could cause the

human metabolism to fail in the myriad of ways necessary

to result in an advanced, systemic autoimmune disease is

increasingly recognized as an outdated 19th century con-

cept. The postulates of Koch are no longer relevant in the

era of the metagenome. Brock contends in his proﬁle of

Koch that attempts to rigidly apply Koch’s postulates to

the diagnosis of viral diseases may have signiﬁcantly im-

peded the early development of the ﬁeld of virology.50

The same can be said for the ﬁeld of bacteriology, where

the Postulates have long impeded researchers from con-

sidering that the genomes of many diﬀerent bacteria and

other pathogens interact together to cause the range of

symptoms we associate with autoimmune diagnoses.

Towards a more nuanced view of the human

microbiota

In New science of metagenomics: revealing the secrets of our

microbial planet, the National Research Council writes,

"The billions of benign microbes that live in the human

gut help us to digest food, break down toxins, and ﬁght

oﬀ disease-causing microbes."51 While certain compo-

nents of the microbiota clearly aid humans in these and

other ways, strictly classifying microbes as either com-

mensal or pathogenic may suggest too categorical a dis-

tinction. Emerging research suggests that bacteria are no

more "good" or "bad" than their human counterparts, par-

ticularly when a commensal microbe can easily acquire a

plasmid or virulence factor from another microbe. Accord-

ing to Fredricks and Relman, “The mobile nature of

virulence-associated gene islands, transported between

bacteria via plasmids or phages, creates the potential for

acquired virulence in previously innocuous microbes."52

In September 2009, Malcolm Casadaban, an infectious

disease researcher at University of Chicago, died suddenly.

An autopsy showed no obvious cause of death except

Yersinia in his bloodstream. Dr. Casadaban, an Associate

Professor at the University, was studying the bacteria to

create a better vaccine for plague. Yet Casadaban was

working with a strain of Yersinia that was supposed to be

less virulent than those strains considered lethal. Re-

searchers postulated that there must have been some-

thing unusual about the bacterium that caused it to be

dangerous, such as a mutation. The so-called "innocuous"

strain of Yersinia may have acquired a plasmid or gene

that endowed it with newfound virulence.

Acquired virulence via horizontal gene transfer has been

studied in anthrax. While Bacillus anthracis -- which causes

fatal poisoning -- and B. cereus, which causes non-lethal

opportunistic infections, are generally classiﬁed as sepa-

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rate bacterial species, Hoﬀmaster discovered a B. cere-

us mutant that also causes a deadly form of pneumonia.

Analysis revealed that the B. cereus mutant (B. cere-

us G9241) had acquired a plasmid with 99.6% sequence

homology to pX01 - B. anthracis' most virulent, toxin-

encoding plasmid. Indeed, B cereus G9241 killed mice

more quickly than B. anthracis. B. cereus G9241 was

deemed the product of horizontal gene transfer, causing

Hoﬀmaster to note that, depending on the extent of hori-

zontal gene transfer, nature could produce an unlimited

number of variations and combinations of any given

pathogen.

The distinction between commensalism and pathogenic-

ity is further blurred by host-speciﬁc factors. For example,

if a species of bacteria aids in the metabolism of carbohy-

drates from the human intestinal tract, the presence of

the microbe in the intestines of famine victims could save

lives. However, in many Western countries, where rates of

obesity are rising at an alarming pace,53

the same microbe

might contribute to excess weight gain.

Returning to the gene/disease network discussed above,

the ACE gene is related to myocardial infarction, renal tu-

bular dysgenesis, Alzheimer's, the progression of SARS,

diabetes mellitus, and sarcoidosis. However, Lactobacillus

and Biﬁdobacteria - species of bacteria considered to be

innocuous or "friendly" are capable of creating a number

of peptides that downregulate expression of ACE.54 These

species of bacteria are added to many of our dairy prod-

ucts and are clearly present in the human body. Yet by

altering the expression of ACE these "friendly bacte-

ria" may well aﬀect the progression of several autoim-

mune and chronic inﬂammatory diseases, albeit in ways

not yet fully understood.

Pathogens alter the expression of human genes

and receptors

Intracellular components create myriad metabolites that

can interfere and alter the correct transcription of human

proteins. Some of these metabolites can also disrupt cellu-

lar repair mechanisms - resulting in the accumulation of

“junk” (e.g. proteins, enzymes, mRNA, etc.) in the cytosol.

For example, Machado et al. reported that Helicobacter

pylori impairs central DNA repair mechanisms, inducing a

transient mutator phenotype, rendering gastric epithelial

cells vulnerable to the accumulation of genetic

instability.55 If the accumulation of errors can exceed the

capacity for cellular repair, such dysregulation not only

has the potential to drive autoimmune processes, but can

result in early senescence,56 apoptosis,57, 58 or cancer.

One of the ways in which pathogens survive is by dys-

regulating the activity of several of the body's key nuclear

receptors. The ability of a number of pathogens to dys-

regulate the Vitamin D Receptor (VDR) - a type 1 nuclear

receptor - provides an excellent example of how microbes

alter human gene expression so as to gain a survival ad-

vantage. Many of the nuclear receptors play a critical role

in regulating immune activity and hormonal expression.

The VDR expresses at least 913 genes, many connected to

autoimmune conditions and cancers. The receptor also

regulates expression of several families of key antimicro-

bial peptides, including cathelicidin and the beta-

Defensins. These play a vital role in allowing the innate

immune system to target intracellular pathogens. For ex-

ample, vitamin D-mediated human antimicrobial activity

against Mycobacterium tuberculosis is dependent on the

induction of cathelicidin.59 The VDR also transcribes Toll-

like-receptor 2 (TLR2), which recognizes bacterial polysac-

charides.

The TACO gene, when expressed, inhibits mycobacterial

entry as well as survival. Mycobacterium tuberculosis (Mtb)

downregulates the VDR, and thus expression of TACO, in

order to survive. Xu et al. showed that the VDR was down-

regulated 3.3 times in monocytic cell lines infected with

Mtb.60 Borrelia, as assessed by BeadChip microarray, has

been shown capable of downregulating VDR activity by a

factor of 50 fold, with lysed Borrelia downregulating the

receptor by a factor of 8.61 We have previously shown that

at least one bacterial metabolite produced by gliding

bioﬁlm bacteria is also a strong VDR antagonist.62

The HIV

“tat” protein binds to the VDR in order to use this receptor

to recognize its Long Terminal Repeat (LTR) promoter

region.63 Thus, tat takes over the human VDR in order to

transcribe HIV's own genome, so the HIV LTR can be rec-

ognized and express new HIV RNA. Tat also recruits his-

tone acetyltransferase activity, including the CREB binding

protein (CBP)/p300 complex, to acetylate the HIV LTR

promoter region.64

Proal et al.: Autoimmune disease and the human metagenome 9

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Slowing the ability of the VDR to express elements of the

innate immune function is such a logical survival mecha-

nism that it is almost certain that other less studied com-

ponents of the microbiota would have also evolved ways

to dysregulate the VDR, and the other nuclear receptors

orchestrating the innate immune response. Eukaryotic

cells respond to the presence of the microbiota by activat-

ing signaling cascades such as the NF-kappaB pathway.

Induction of such pathways leads to the upregulation of

gene expression mediating pro-inﬂammatory and anti-

apoptotic eﬀector proteins. Thus, in order for pathogens

(and potentially, symbionts) to continue their life cycle, it

is necessary to evade or repress these cellular responses.

This is especially true because acquisition of resistance to

antimicrobial peptides by a sensitive microbial strain is

surprisingly improbable. Furthermore, the extension of

human life during the past century now oﬀers additional

opportunity for microbes to evolve their specialization in

order to survive in man.

Indeed, Yenamandra et al. recently showed that Epstein-

Barr Virus also slows VDR activity.65 Infection of human B

cells with EBV induces metabolic activation, morphologi-

cal transformation, cell proliferation and eventual immor-

talization by altering the expression of a number of key

nuclear receptors. The team found that the expression of

12 nuclear receptors was downregulated in the longer-

lasting, younger lymphoblastoid cells. Among them was

the VDR and the Estrogen Receptor Beta (ERB), both

downregulated by a factor of about 15 times (Figure 2).

EBV is found in many common chronic disease states. In-

deed, EBV has been detected in a subset of patients with

nearly every autoimmune diagnosis, although it has rare-

ly been detected in 100% of patients with any given con-

dition. In some cases, infection with the virus is described

as a "precipitating factor" for autoimmune disease. That

EBV downregulates VDR and ERB expression may explain

this phenomenon. If a patient acquires EBV, the virus

slows innate immune activity to the point where the en-

dogenous microbiota can become dominant.

This is particularly true because, in addition to reducing

expression of cathelicidin and beta-Defensin, VDR dys-

regulation opens a number of other pathways that also

inﬂuence immune activity and hormonal regulation.

Blockage of the VDR prevents transcription of CYP24A1,

an enzyme that normally breaks down excess levels of the

active vitamin D metabolite 1,25-dihydroxyvitamin-D

Proal et al.: Autoimmune disease and the human metagenome 10

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Figure 2. Nuclear receptors mRNA expression is downregulated upon infection of B-cells with EBV 65

(1,25-D). Activation of Protein Kinase A (PKA) by bacterial

cytokines also causes increased production of the enzyme

CYP27B1, resulting in increased conversion of 25-

hydroxyvitamin-D (25-D) into 1,25-D. Both processes re-

sult in a rise in 1,25-D.

High levels of 1,25-D in autoimmune disease have been

conﬁrmed in a clinical setting. Mawer et al. found that

1,25-D levels were particularly elevated in the synovial

ﬂuid surrounding the joints of patients with rheumatoid

arthritis.66 Abreu et al. found that in a cohort of 88 Crohn's

disease patients, 35 patients or 40% had elevated levels of

1,25-D, which the authors deﬁned as above 60

pg/ml.67 Bell noted that patients with tuberculosis, pneu-

monia, AIDS, disseminated candidiasis, leprosy, rheuma-

toid arthritis, silicone-induced granulomas, Wegerner’s

granulomatosis, Hodgkin’s disease, lymphoma, histocytic

lymphoma, T-cell leukemia, plasma cell granuloma, leio-

myoblastoma, seminoma, and subcutaneous fat necrosis

all tend to manifest with higher than normal levels of

1,25-D.68 Blaney et al. found that of 100 patients with vari-

ous autoimmune diagnoses, 85 percent had 1,25-D above

the normal range (Figure 3).69 Yoshizawa et al. reported

that in VDR knockout mice, a circumstance that closely

mimics extreme VDR dysregulation, 1,25-D levels increase

by a factor of ten.70 However, understanding 1,25-D's role

in various inﬂammatory disease states is complicated by

the fact that most researchers determining vitamin D

status test subjects only for levels of the inactive vitamin

D metabolite, 25-D.

In silico research indicates that 1,25-D has a high aﬃnity

for, not just the VDR, but many of the body’s other nuclear

receptors.71 This suggests that at high concentrations it

will displace their exogenous ligands. Those receptors af-

fected by elevated 1,25-D include alpha thryoid, beta thy-

roid, the glucocorticoid (adrenal) receptor, and the pro-

gesterone receptor (Figure 4). For example, 1,25-D has a

very high aﬃnity for the thyroid beta, suggesting that it

can displace T3 and T4 from the binding pocket (Table

1).71

If 1,25-D prevents T3 from activating thyroid beta, then

genes with thyroid beta promoters will be less energeti-

cally transcribed. This would result in thyroid disease and

Proal et al.: Autoimmune disease and the human metagenome 11

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Figure 3. 25-D vs. 1,25-D in a cohort of 100 autoimmune patients 69

explain why increasing levels of thyroid hormone are nec-

essary to maintain thyroid homeostasis as chronic disease

progresses. Furthermore, since the functions of type 1 nu-

clear receptors are largely interdependent, if transcription

by thyroid beta is dysregulated, system wide transcription

is also aﬀected.

This leads to disruption of system-wide anti-microbial

peptide (AMP) production. Just as the VDR expresses

cathelicidin and beta-Defensin, other nuclear receptors

also express AMPs. Brahmachary et al. have shown that

the Glucocorticoid receptor, the Androgen receptor, and

the Vitamin D Receptor, are respectively in control of 20,

17 and 16 families out of the 22 analyzed.72 Thus, dysregu-

lating VDR activity yields ﬂow-on eﬀects that potentially

disable the bulk of the body's antimicrobial peptides. A

patient aﬀected in this manner would become increas-

ingly immunocompromised, allowing disease-causing

components of the microbiota to proliferate with even

greater ease.

This supports a disease model in which key components

of the microbiota responsible for autoimmune condi-

tions gradually shut down the innate immune response

over a person's lifetime as bacteria, and other pathogens,

incrementally accumulate into the microbiota. Crohn’s

disease is already characterized by diminishing functional

antimicrobial activity, particularly when it comes to ex-

pression of cathelicidin and the beta-Defensins.73

Eventu-

ally, genes from the accumulating microbial metagenome

may instigate a clinical disease symptomology, such as

one of the autoimmune diagnoses, or simply drive the

inﬂammation associated with the aches and pains of ag-

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Table 1. Afﬁnities of Native Ligands and 1,25-D for Various

Nuclear Receptors

Nuclear receptor

Native

ligand

Native

ligand (Kd)

1,25-D

(Kd)

α-Thyroid

T3

7.20

8.41

β-Thyroid

T3

7.18

8.44

Glucocorticoid

Cortisol

7.36

8.12

Androgen

Testosterone

7.38

8.05

Progesterone

7.53

8.09

Figure 4. The Thyroid-alpha nuclear receptor and T3, its native ligand [PDB:2H77], with the bound conformation

of 1,25-D superimposed. Since the XSCORE Kd for 1,25-D is 8.4, and for T3 is 7.2, it is apparent that 1,25-D is capa-

ble of displacing T3 from binding to key receptor residues (shown here are Arg228, Asn179, Gly290, Leu292,

Leu276, Ser277, Thr275, Ala263, Leu287, Ala180, Phe218 and Arg162).71

ing. Indeed, the lifelong accumulation of an increasingly

diverse microbiota directly correlates with an age-related

increase in diseases and symptoms associated with in-

ﬂammation. The term "inﬂammaging" has been coined to

explain "the now widely accepted phenomenon that ag-

ing is accompanied by a low-grade chronic, systemic up-

regulation of the inﬂammatory response, and that the un-

derlying inﬂammatory changes are common to most age-

associated diseases.”74

Because 1,25-D is expressed in the human cycling en-

dometrium and rises by 40% during early pregnancy,

women may be disproportionately aﬀected by the poten-

tial drop in AMP expression associated with VDR

dysregulation.75 This implies that females may more easily

accumulate a more diverse microbiota than their male

counterparts, which could help explain why women suﬀer

from a higher risk of most autoimmune diagnoses.

Successive infection and variability in disease

onset and presentation

The makeup of a person’s microbiota is unique: hu-

mans may share as li t tle as 1% o f the same

species.76 Given that the human microbiome may play the

principal causative role in autoimmune disease, it may not

be by accident that the uniqueness with which patients'

autoimmune disease symptoms develop parallels the in-

credible variability of the human microbiome. Tradition-

ally, diseases have been understood to be discrete and

have their own respective and distinct pathologies. Hence

the emphasis on diagnosis. But, if the spectrum of auto-

immune disease were driven by a common factor –

namely a person's microbial inhabitants – variability in

disease could be explained by accounting for how the

human microbiota accumulates and develops in any one

person. Enck et al. recently analyzed fecal ﬂora of stool

samples from 35,292 adults whose ages ranged from 18 to

96 years of age in order to gauge the relative abundance

and composition of various bacterial species over time.16

He found that while the number of bacteria in the fecal

microbiota remained stable with age, the composition of

the microbiota diversiﬁed as subjects became older, with

the oldest subjects measured (over 60 years of age) repre-

senting the most profound changes. Older subjects were

much more likely to have higher prevalence of microbes

associated with chronic disease such as Enterococcus and

E. coli.

A number of microbes that slow immune activity have

already been identiﬁed indicating that bacteria/viral-

driven suppression of innate immune activity may occur

on a much larger scale than previously imagined. Each

pathogen that decreases immune activity makes it easier

for the host to pick up other pathogens, which themselves

may further slow immune activity, creating a snowball

eﬀect. This process is known as successive infection and

oﬀers us a framework for understanding how not only

diseases of the gastrointestinal tract develop, but also any

number of other autoimmune and inﬂammatory diseases.

As human genes are upregulated or downregulated by

acquired components of the microbiota, the body shifts

farther and farther away from its natural state of homeo-

stasis. Infected cells increasingly struggle to correctly pro-

duce human metabolites in the presence of numerous

proteins and enzymes being created by the pathogenic

genomes.

The ease with which a person acquires a pathogen from

the environment, or from another person, depends largely

on the state of their immune system. Those people who

harbor low pathogenic loads and still have an active in-

nate immune system, could be expected to kill the acute

and chronic pathogens they encounter. Conversely, those

people with a compromised immune system will accumu-

late pathogens over time. We have previously discussed

how VDR dysfunction, along with adrenal and androgen

dysfunction, predispose to a weakened innate immune

system, but there are many other factors in play. For ex-

ample, Bukholm and team found that when the measles

virus infects cell cultures, those cells are more susceptible

to a secondary bacterial invasion.77

Stress has also been shown to impede immune function,

by inhibiting natural killer cell activity, lymphocyte popu-

lations, lymphocyte proliferation, antibody production

and reactivation of latent viral infections.78 Already identi-

ﬁed consequences on health include delayed wound heal-

ing, impaired responses to vaccination and development

and progression of cancer.79 Depending on the variety of

stressful events that occur over a lifetime, people may be

more susceptible to picking up microbes at diﬀerent

times. The immune response could be expected to be par-

Proal et al.: Autoimmune disease and the human metagenome 13

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

ticularly weak after traumatic events such as surgery, a car

accident or even a pregnancy.80

People accumulate microbiota-altering pathogens in myr-

iad diﬀerent ways, the most obvious being social contact.

People in close proximity, particularly spouses and chil-

dren inevitably pick up components of each other's

microbiomes.81 Healthcare workers have a higher rates of

certain autoimmune and inﬂammatory conditions includ-

ing breast cancer and malignant melanoma.82 Merely

shaking hands causes the transfer of numerous

microbes.83

Genomic analysis of the bacteria on the hands

of students leaving an exam room contained 332,000 ge-

netically distinct bacteria belonging to 4,742 diﬀerent

species. Forty-ﬁve percent of the species detected were

considered rare. This marked a hundred-fold increase in

the number of bacterial species detected over previous

studies that had relied on purely culture-based methods

to characterize the human hand microbiota.

Obesity is not currently accepted as an autoimmune con-

dition, but Christakis and Fowler recently used quantita-

tive analysis of a densely interconnected social network to

conclude that obesity is transmitted among people.84 A

person’s risk of becoming obese increases by 57% if they

have a friend who becomes obese, and by 37% if their

spouse becomes obese. While, as the team concludes,

people may mimic the behavior of friends or family in

ways that could cause them to gain or lose weight, it is

also possible that the close proximity among many of the

subjects in the study would have allowed them to directly

exchange microbes. Since the composition of bacteria in

the gut has, in several instances, been linked to the devel-

opment of obesity85 – perhaps, in some cases, obesity is

literally contagious. It seems likely the same could be said

for any autoimmune condition with an infectious etiology.

In some cases, pathogens may be acquired in the womb,

particularly if the mother already suﬀers from one or more

autoimmune or inﬂammatory diagnoses. Similarly, bacte-

rial species including Staphylococcus epidermidis, Strepto-

coccus viridans, E. coli, Staphylococcus aureus, Streptococcus

faecalis, Proteus and others have been detected in

sperm.86 Mycobacterium tuberculosis and inﬂuenza HSN1

have been shown to cross the placental barrier. Already

implicated in implantation failure, spontaneous abortion,

and preterm birth, infection with Shigella is now proposed

to cause endometriosis.87 DiGiulio studied ribosomal DNA

(rDNA) of bacteria, fungi and archaea from amniotic ﬂuid

of 166 women in preterm labor with intact membranes.

Fifteen percent of subjects harbored microbes that to-

gether belonged to 18 diﬀerent taxa - including Sneathia

sanguinegens, Leptotrichia amnionii and an unassigned,

uncultivated, and previously uncharacterized bacterium.

A positive PCR was associated with histologic chorioam-

nionitis and funisitis. The correlation between positive

PCR and preterm delivery was 100%.

Pathogens can also pass from mother to child during

breast-feeding. For example, Human papillomavirus type

16 (also called high-risk HPV-16), which has been linked to

cervical cancer, has been detected in human breast milk

collected during the early period after a woman delivers

her baby.88 Pathogens can also be transmitted from per-

son to person through bodily ﬂuids released during

coughing, sneezing and other intimate contact and are

found nearly everywhere in our environment. For exam-

ple, non-tuberculosis Mycobacteria and other opportunis-

tic human pathogens are enriched to high levels in many

showerhead bioﬁlms, >100-fold above background water

contents. Catheters used to treat urinary tract infections

and other conditions have, in some cases, been shown to

harbor copious amounts of bioﬁlm.

Early infections predispose a person to later

chronic disease

Most of the bacteria implicated in autoimmune disease

are slow-growing pathogens whose eﬀects will take dec-

ades to manifest.89 In this sense, bacteria acquired earlier

in life can alter the ultimate microbiota in ways that may

not be recognized for decades. According to Merkler et al.,

“In genetically susceptible individuals, early childhood

infections seem to predispose them to [such disease as]

multiple sclerosis or type 1 diabetes years or even dec-

ades before clinical onset.”90 A 2006 report by the Centers

for Disease Control (CDC) echoes this sentiment: “A per-

son’s age at the time of infection—from intrauterine or

perinatal (the time period surrounding birth), through

childhood and adolescence, to adulthood and the elder

years—may further inﬂuence the risk for chronic out-

come. For example, perinatal herpes virus infection dra-

matically increases the risk of developing adult or pediat-

Proal et al.: Autoimmune disease and the human metagenome 14

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ric chronic liver disease. Recurrent infections or perhaps

serial infections with certain agents might also determine

a person’s risk for chronic outcome.”91

Thus, while Medicine generally assumes that once a per-

son has recovered from an acute illness, they return to a

state of complete health – so-called "sterilizing immunity"

– in truth, the long-term consequences of acute infection

are somewhat poorly understood. Newborns who harbor

certain types of bacteria in their throats, including Strep-

tococcus pneumoniae and Haemophilus inﬂuenzae are at

increased risk for developing recurrent wheeze or asthma

early in life.92 Approximately two-thirds of patients with

Guillain-Barré syndrome, a suspected autoimmune condi-

tion, have a history of an antecedent respiratory tract or

gastrointestinal infection.28 Prenatal infections such as

rubella, inﬂuenza, and toxoplasmosis are all associated

with higher incidence of schizophrenia - with the children

of those mothers exposed to inﬂuenza in the ﬁrst trimes-

ter of gestation showing a seven-fold increased risk of

schizophrenia.93 Reactive arthritis (Reiter's syndrome) is

classically seen following infection with enteric pathogens

such as Yersinia, Salmonella, Campylobacter and

Shigella.94 Acute gastroenteritis, resulting from infection

with the same pathogens, causes approximately 6-17% of

patients to develop chronic irritable bowel syndrome.

In an especially provocative experiment, a team including

Doron Merkler and Nobel Laureate Rolf Zinkernagel in-

jected cytomegalovirus (CMV) into the brains of mice that

were only a few days old.90

The innate immune systems of

the mice were able to eliminate CMV from most of the

tissues except for those of the central nervous system. As

a result, the virus persisted in the brains of the mice. Later

in life, when the same mice were challenged by infection

with a similar virus, they developed a condition resem-

bling a type of autoimmune disease and died. The team

referred to this concept as "viral déjà vu.”

Incidents of food poisoning also point to unresolved fea-

tures of acute infections. Siegler et al. noted that 10% of

people who suﬀered from E. coli food poisoning later de-

veloped a relatively infrequent life-threatening complica-

tion called hemolytic uremic syndrome (HUS) where their

kidneys and other organs fail.95 According to the study,

10-20 years after patients recover, between 30-50% of E.

coli survivors will have some kidney-related problem,

conditions that include high blood pressure caused by

scarred kidneys, slowly failing kidneys, or even end-stage

kidney failure requiring dialysis.

Microbes can also be transmitted by donation of blood,

bone marrow transplants, or organ donation, which, if

pathogenic, can greatly disrupt the composition of the

microbiota over time. The term "donor-acquired sarcoido-

sis" refers to the development of sarcoidosis in presuma-

bly naïve (non-sarcoidosis) transplant recipients who have

received tissues or organs from donors who were not

known or suspected to have active sarcoidosis.96

Murphy

studied over 8,500 people in the United Kingdom who

underwent heart surgery between 1996 and

2003.97 Patients who had received red blood cell transfu-

sions were about three times more likely to suﬀer a heart

attack or stroke and were at a higher risk for infection, re-

admission to hospital, and death compared with heart

patients who did not receive blood. The risks associated

with blood transfusions were not inﬂuenced by a patient’s

age, hemoglobin levels or the extent of their disability at

the time of transfusion. Writing in the journal Circula-

tion, Murphy et al. concluded: “Red blood cell transfusion

appears to be harmful for almost all cardiac surgery pa-

tients and wastes a scarce commodity and other health

service resources.”97

Comorbidity

Thus the catastrophic failure of the human metabolism

we see in autoimmune disease – which at ﬁrst glance ap-

pears so diverse and so diﬀerent among diﬀerent diagno-

ses – appears to be due to a single underlying mecha-

nism: a ubiquitous microbiota, much of which has evolved

to persist in the cytoplasm of nucleated cells. What diﬀers

among individuals as they gradually acquire a unique mi-

crobiota over time is the virulence, location, and combina-

tion of those pathogenic species. The high rate of comor-

bidity among inﬂammatory diagnoses98 lends support for

this explanation. Such comorbidity between seemingly

unrelated diseases cannot be explained by laws of aver-

age – the risk of autoimmune disease is not evenly dis-

tributed. Figure 5 demonstrates the degree of comorbid-

ity seen among various inﬂammatory diagnoses. Each

“spoke” represents a study from PubMed which has dem-

onstrated a signiﬁcant statistical relationship between

Proal et al.: Autoimmune disease and the human metagenome 15

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

patients suﬀering from one inﬂammatory disease and the

next.

In the case of multiple sclerosis, Barcellos et al. identiﬁed

coexisting autoimmune phenotypes in patients with mul-

tiple sclerosis from families with several members with the

disease and in their ﬁrst-degree relatives.99

A total of 176

families (386 individuals and 1107 ﬁrst-degree relatives)

were examined for a history of other autoimmune disor-

ders. Forty-six (26%) index cases reported at least one co-

existing autoimmune disorder. The most common were

Hashimoto’s thyroiditis (10%), psoriasis (6%), inﬂamma-

tory bowel disease (3%), and rheumatoid arthritis (2%).

One hundred and twelve (64%) families with a history of

multiple sclerosis reported autoimmune disorders (ex-

cluding multiple sclerosis) in one or more ﬁrst-degree

relatives. Hashimoto’s thyroiditis, psoriasis, and inﬂamma-

tory bowel disease were also the most common diagno-

ses occurring in these family members. Such high rates of

comorbidity support a model for autoimmune conditions

in which no two people with the same diagnosis ever de-

velop the exact same disease presentation; the interac-

tions between an individual's genome and their unique

metagenome are so varied that they are rarely identical.

Note that Figure 5 suggests that patients with autoim-

mune diagnoses are also much more likely to suﬀer from

mental conditions such as depression and anxiety. In-

creasing clinical evidence, including that from our own

study100, conﬁrms the involvement of microbiota in neu-

rological disease states. This suggests that both autoim-

mune and neurological diagnoses, which are currently

balkanized into separate medical specialties, most proba-

bly result from the same underlying dysregulation of mi-

crobial populations.

Autoimmune and inﬂammatory conditions also suﬀer

from specialty delineation. For example, VDR dysregula-

tion does not just impact the autoimmune disease state.

Researchers have reported epigenetic repression of VDR

Proal et al.: Autoimmune disease and the human metagenome 16

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

Figure 5. Comorbidities among common inﬂammatory diseases. Each “spoke” of this wheel represents a pub-

lished study appearing in MEDLINE, which shows a signiﬁcant statistical relationship between one disease and

another

gene expression and activity in choriocarcinoma cell

lines.101 Furthermore, the VDR expresses genes involved in

both autoimmune and inﬂammatory processes. It tran-

scribes insulin-like growth factor (IGFBP-3),102 which inﬂu-

ences the development of diabetes, yet also expresses

Metastasis Suppressor Protein 1 (MTSS1), which plays a

vital role in repressing the cell cycle and promoting apop-

tosis in cancerous cells.102 Drawing a line between auto-

immune and inﬂammatory disease makes these and other

common mechanisms harder to recognize and study.

Causation versus association

If most autoimmune and inﬂammatory conditions do in-

deed arise from the same underlying disease process,

then we must re-examine some of the cause and eﬀect

relationships postulated to exist among inﬂammatory

conditions. For example, it is commonly believed that

obesity is a causative factor in the development of

diabetes.103 In fact, patients with type 2 diabetes are so

likely to become morbidly obese that the two conditions

are sometimes collectively referred to as "diabesity."104

Obesity has been tied to microbial composition in the

gut105, the result of a microbial process. Roesch et

al. found that the onset of type 1 diabetes was tied to the

presence of speciﬁc bacteria in the murine

gut.106 Additionally, at least one microbial species, Strep-

tomyces achromogenes, secretes a substance, streptozocin,

which can directly induce type 1 diabetes.107 The diabetes

disease process would also make it substantially harder

for the immune system to regulate microbial gut compo-

sition. In particular, species that are extremely eﬀective at

extracting calories from food may thrive while their in-

nocuous counterparts may ﬁnd themselves

out-competed. The expression of hormones that regulate

appetite, such as leptin or ghrelin could also become dys-

regulated by the bacterial microbiota.108 For example, H.

pylori infection leads to a decrease in circulating ghrelin

through a reduction in ghrelin-producing cells in the gas-

tric mucosa.109 In some cases, this could cause weight gain

even in the absence of excess calorie consumption.110 In

light of the above, obesity and diabetes might better be

described as developing simultaneously. Treatments

aimed at addressing those underlying factors contribut-

ing to both disease states might well prove the most ef-

fective.

The same dichotomy is found in other sets of parallel con-

ditions such as tooth decay and dementia, rheumatoid

arthritis and uveitis, high cholesterol and heart disease,

and others. It is far more likely that both conditions arise

from a common metagenomic microbiota than that one

condition is causal for the other.

Microbial interaction and disease

One of the more striking characteristics of non-obese dia-

betic (NOD) mice is that exposure to Mycobacteria can

prevent the onset of diabetes while precipitating lupus in

the same animal.111, 112 While this phenomenon is diﬃcult

to interpret by studying the murine genome alone, it may

help to consider the murine metagenome. If, as in hu-

mans, the murine metagenome causes disease as it ac-

cumulates over time, then the interactions between vari-

ous microbial species may be telling. Even within the con-

text of the ultimate example of symbiotic behavior, the

bioﬁlm, bacteria have been shown to compete with one

another, sometimes even "cheating" to do so.113 We would

not have many antibiotics if it weren't for competition

among bacterial species. For example, the early tetracy-

cline antibiotics were derived from species of Streptomy-

ces, and are toxic to a number of its competitors.

With the NOD mice, introduction of a new species of bac-

teria into the microbiota, Mycobacteria, may alter the mi-

crobiota in such a way as to wipe out, or at least diminish,

the diabetes disease state. At the same time, the microbi-

ota allows lupus to proliferate or dominate. Similar com-

petition between microbes may also explain why lupus

has been shown to inhibit the development of malaria

(Plasmodium falciparum).114

Autism, an inﬂammatory condition that has been associ-

ated with several unique microbial populations,115 may

have a comparable dynamic at work. In children diag-

nosed with autism spectrum disorder, fever associated

with intercurrent bacterial or viral infections - such as up-

per respiratory infections - has been shown to temporarily

decrease aberrant behavior such as irritability and inap-

propriate speech.116

Gastric surgery invariably alters the composition of the

gastrointestinal microbiota. DePaula et al. found that after

Proal et al.: Autoimmune disease and the human metagenome 17

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

39 diabetic type 2 patients in Brazil underwent bariatric

surgery, all subjects no longer required insulin therapy.117

All subjects also experienced normalization of their cho-

lesterol levels, 95.8% had their hypertension controlled,

and 71% achieved targeted triglyceride levels. This corre-

lates with data showing that the intestinal bacterial popu-

lations of normal weight individuals, morbidly obese indi-

viduals, and people who have undergone gastric bypass

surgery are distinctly diﬀerent. For example, Firmicutes

were dominant in normal-weight and obese individuals

but signiﬁcantly decreased in post-gastric-bypass indi-

viduals, who had a proportional increase of

gammaproteobacteria.118

Other microbial interactions can alter the pathogenicity of

one or more species involved. The pathogenic potential

of Helicobacter hepaticus in a mammalian colitis model is

altered by the presence of diﬀerent strains of Bacteroides

fragilis. When the bacterial polysaccharide PSA is ex-

pressed on the microbial cell surface of B. fragilis, it sup-

presses pro-inﬂammatory interleukin-17 production to H.

hepaticus.119

Hoﬀman et al. found that when the bacterial

species Pseudomonas. aeruginosa and S. aureus were in-

cubated together, P. aeruginosa created a protein, HQNO,

which protected S. aureus from eradication by commonly

used aminoglycoside antibiotics such as tobramycin.120

Also, in cases of P. aeruginosa and S. aureus co-infection in

the presence of HQNO, small-colony variants of S. aure-

us are selected for, making S. aureus more diﬃcult for the

immune system to target. While we are far from under-

standing the full nature of these microbial interactions, it

is clear that a microbiota constantly evolves so that the

symptoms of any given disease are seldom static.

Familial aggregation

The common disease-common variant hypothesis sug-

gests that chronic diseases are the product of anywhere

from one to thousands of disease-causing alleles. The

HapMap single nucleotide polymorphisms (SNP)-

cataloging project has identiﬁed over 3.1 million SNPs,

with many more expected to be found as the project con-

tinues. However only a fraction of these SNPs confer any

more than a minimal statistically increased risk for

disease.121

For example, in cancer, for nearly all regions

conclusively identiﬁed by genome wide association stud-

ies (GWAS), the per allele eﬀect sizes estimated are less

than 1.3. While over 85 regions have been conclusively

associated in over a dozen diﬀerent cancers, no more than

ﬁve regions have been associated with more than one

distinct cancer type.121 According to Stephen Chanock of

NIH, “Nearly every candidate SNP [associated with cancer]

has failed in the long run – maybe ﬁve or six are real by

rigorous standards.”(Personal communication)

There appear to be factors at work other than just Mende-

lian inheritance. The increased risk of chronic disease

amongst non-relations in close proximity – so-called "case

clusters" – strongly implies an infectious dynamic at work.

The evidence that the autoimmune disease sarcoidosis is

communicable is particularly strong. A study of 215 sar-

coidosis patients found that ﬁve husband-and-wife cou-

ples both had the disease - a rate 1,000 times greater than

could be expected by chance.122 The NIH ACCESS research

team also noted that the risk for sarcoidosis increased

nearly ﬁve-fold in parents and siblings of people with the

disease. A case-controlled study of residents of the Isle of

Man found that 40% of people with sarcoidosis had been

in contact with a person known to have the disease, com-

pared with 1 to 2% of the control subjects.123 Another

study reported three cases of sarcoidosis among ten ﬁre-

ﬁghters who apprenticed together.124

The literature contains many examples of unexpected fa-

milial associations among seemingly distinct disease pa-

thologies. For example, a 2008 study of parents of children

with autism found they were more likely to have been

hospitalized for a mental disorder than parents of control

subjects, with schizophrenia being more common among

case mothers and fathers compared with respective con-

trol parents.125 In the case of schizophrenia and autism,

both have been associated with prenatal viral

infection.126 While a fetus can acquire these and many

other pathogens directly, successive infection dictates

that as children age they will manifest with inﬂammatory

symptoms that may diﬀer from those of their parents. Ma-

jor factors that would inﬂuence the development of a dis-

crete inﬂammatory diagnosis include the mix of species

acquired, the sequence in which the pathogens are ac-

quired, the subsequent changes in gene expression

caused by the pathogens, and the profound eﬀect on the

body’s proteins, enzymes and metabolites caused by

these changes. Because the adaptive immune response in

Proal et al.: Autoimmune disease and the human metagenome 18

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

infants takes several weeks to develop, infants are particu-

larly prone to picking up pathogens during the ﬁrst weeks

of life.92 Such pathogens could be acquired from any fam-

ily or friends in contact with the child, especially the

grandparents, who probably harbor some of the highest

pathogenic loads. Palmer et al. found that infants pick up

many of the species that make up their gut ﬂora from fam-

ily members within just a few weeks of birth, suggesting

that non-gut bacteria may easily be acquired during this

time as well.127

Is autoimmune disease predisposition

Mendelian?

Two decades ago, the attention of the research commu-

nity shifted towards a new source in an attempt to explain

the etiology of autoimmune disease: the human genome.

Begun formally in 1990, the U.S. Human Genome Project

was a 13-year eﬀort coordinated by the U.S. Department

of Energy and the NIH. Its primary goal was to determine

the sequence of chemical base pairs that make up DNA

and to identify the genes of the human genome from

both a physical and functional standpoint. A working

draft of the genome was released in 2000 and a complete

version in 2003, with further analyses yet to be completed

and published.128

Meanwhile, the private company Celera

Genomics conducted a parallel project.129

Early in the aftermath of the sequencing of the human

genome, many geneticists advocated the common

disease-common variant hypothesis, expressing certainty

that the ﬁeld would quickly determine genetic haplotypes

that would correlate with and explain the bulk of chronic

diseases. Dr. Francis Collins' 2001 statement was typical: "It

should be possible to identify disease gene associations

for many common illnesses in the next 5 to 7 years."130

Researchers hoped that by dissecting the human genome,

patients could be informed that they had "the gene" for

breast cancer, sarcoidosis, rheumatoid arthritis, or any of

the other autoimmune diagnoses. Targeted gene thera-

pies could then be developed to eﬀectively eradicate

these conditions.

It may be too early to call human genomic research an

unqualiﬁed failure,131

but it is diﬃcult to ignore a lack of

utility in identiﬁcation of disease. Recently, the limited

progress in the genetic analysis of common diseases has

begun to be acknowledged.132, 133

Certainly there have

been no widely successful gene therapies to date, and

genome-driven personalized medicine has yet to live up

to its early promise. To identify what some researchers

refer to as the "missing heritability," geneticists have pro-

posed GWA studies with historically unprecedented sam-

ple sizes. In the last year, researchers have publicly con-

templated "daunting" sample sizes exceeding 500,000

subjects in concert with studies that would be conducted

over periods as long as 45 years.134

Ewald et al. argue that evolutionary forces that would

cause a serious disease to be weeded from the population

would also cause those people whose immune systems

are prone to self-attack to be eliminated from the

population.135 An exception would occur if the disease

oﬀers a survival advantage. For example, the genetic dis-

orders cystic ﬁbrosis may confer resistance to

tuberculosis.136 The Mendelian disorder sickle cell anemia

is common in tropical countries because it confers resis-

tance to malaria. With malaria, researchers can quantify

the rate by generation at which the gene for sickle cell

anemia is dropped from the population in the absence of

an evolutionary advantage – as is the case when people

migrate away from malaria-infested areas. However, no

autoimmune diagnosis has been shown to confer any sort

of beneﬁcial survival trait. Under these circumstances, one

would expect any faulty gene or network of genes associ-

ated with an autoimmune condition to be selected

against, especially since many autoimmune conditions

strike during the reproductive years. Chronic diseases

have existed for thousands of years with manifestations of

both arteriosclerosis137 and cardiac disease observed in

mummies of ancient Egypt.138 Ötzi the Neolithic Iceman

who lived around 3300 BC had arthritis, allowing ample

time for any alleles associated with autoimmune disease

to be eliminated via natural selection.139 Instead, the

prevalence of autoimmune conditions seems to have re-

mained essentially constant until quite recently.

SNPs and autoimmune disease

After noting that amongst his cohort of 31 patients with

abdominal aortic aneurysm, SNPs in the gene BAK1 were

diﬀerent in aortic tissue than in blood samples from the

same patients,140

Gottlieb remarked, "Genome-wide asso-

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This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

ciation studies were introduced with enormous hype sev-

eral years ago, and people expected tremendous break-

throughs. Unfortunately, the reality of these studies has

been very disappointing, and our [own] discovery cer-

tainly could explain at least one of the reasons why." The

conundrum that Gottlieb's study has exposed is that the

human genome appears to vary between the tissue and

plasma compartments. Medicine has always assumed that

human DNA is homogeneous throughout the human

body. We now need to explore the mechanisms whereby

these diﬀerent genetic sequences could arise through

selective pressure in diﬀerent tissues such as would exist if

the tissue harbored a microbiota.

One of the mechanisms proposed for genetic predisposi-

tion states that genetic haplotypes predispose for disease

processes. Because it is a highly polymorphic genomic

region, MHC has served as the preferred axis for studying

susceptibility to immune diseases. Major changes have

been detected within the HLA class I and class II genes

related to various populations across the globe. For ex-

ample, in Type 1 diabetes, the most common haplotype in

the Western world is AH8.1 (HLA-A1-B8 DR3 -SC01). How-

ever, this haplotype is almost nonexistent in the Indian

population, and has been supplanted by the variant

AH8.1v which diﬀers from the Caucasian AH8.1 at several

gene loci.141 Moreover, there are additional HLA-DR3 hap-

lotypes HLA-A26-B8-DR3, HLA-A24-B8 DR3 (AH8.3), A2-B8-

DR3 (AH8.4) and A31-B8-DR3 (AH8.5) that occur largely in

the Indian population alone.

Similarly, the FCRL3-169T-C polymorphism, which is sig-

niﬁcantly associated with rheumatoid arthritis (RA) in East

Asian populations is not associated with RA in Caucasians

of European decent.142 Interestingly, the frequency of the

rs7528684 minor allele associated with FCRL3- varies as

much within each of the two ethnic groups as it does be-

tween them. Furthermore, a recent large case-controlled

study found that FCRL3-169T-C was not signiﬁcantly asso-

ciated with RA in Korean patients.142

Thus, no diagnostic certainty can be obtained by measur-

ing genes on the HLA axis. None of the HLA haplotypes

cause disease 100% of the time and none cause any one

immune disease consistently. Patterns of haplotype varia-

tion are more suggestive of a regional infectious model

rather than a model in which an illness is caused by wide-

spread inherited variation of HLA haplotypes.

Potential systematic errors in the interpretation

of the metagenome

Primers selected for most epidemiological studies are

chosen without consideration for whether they might

amplify DNA from the genomes of any intracellular mi-

crobes. As artist Pablo Picasso once remarked, "Computers

are useless. They can only give you answers." If a software

program fails to make provision for the possibility that a

metagenome might also be present, the chances of a false

positive increase signiﬁcantly during the process of ge-

nomic analysis. Similarities between bacterial and human

genes will likely cause the analysis software to not assem-

ble the genomic data properly. The likelihood of error is

not minuscule as there is growing evidence of molecular

mimicry, homology between bacterial and human pro-

teins. For example, signiﬁcant sequence homology exists

between human carbonic anhydrase II and alpha-carbonic

anhydrase of H. pylori.143 Moreover, the homologous seg-

ments contain the binding motif of the HLA molecule

DRB1*0405. The group A streptococcal carbohydrate anti-

gen N-acetyl-glucosamine is able to cross react with car-

diac myosin.144 Microbes including E. coli, H. pylori, P. ae-

ruginosa, Cytomegalovirus, and H. inﬂuenzae share se-

quence homology with human pyruvate dehydrogenase

complex-E2, which has been tied to the development of

primary biliary cirrhosis.145 The core oligosaccharides of

low-M(r) LPSs of C. jejuni serotypes that are associated

with the development of Guillain-Barré syndrome are

homologous to neural gangliosides.

Before we can be certain that all measured SNPs and HLA

haplotypes are a product of only the human genome and

not the metagenome, researchers must begin to actively

choose PCR primer pairs that are unlikely to amplify mi-

crobial DNA. Primers need to not only be certiﬁed to am-

plify a unique sequence in the human genome, they need

to be certiﬁed as not likely to amplify genes from any of

the thousands of bacterial and viral genomes in the

metagenomic databases. While PCR ampliﬁcation usually

involves more than one stage of genomic selectivity, the

increasing use of arrays of RNA probes increases the likeli-

hood that a fragment of metagenomic RNA will unex-

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This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

pectedly match a probe, and increases the possibility of a

false-positive being signaled for the particular SNP being

sought.

Antibodies in response to microbial DNA

Autoimmune diseases are characterized largely by the

presence of autoantibodies. While autoantibodies were

reported over a century ago, many scientists at the time

were unwilling to accept the possibility that the immune

system attacks its own cells. Ehrlich argued that autoim-

munity was not possible and proposed the theory of hor-

ror autotoxicus to describe the body's innate aversion to

immunological self-destruction by the production of

autoantibodies. Now that humans are understood to be

the product of multiple genomes, increasing evidence

supports Ehrlich's view. When an innate immune system is

forced to respond to a chronic microbiota, the resulting

cascade of chemokines and cytokines will also stimulate

an adaptive response. Antibodies are notoriously poly-

speciﬁc, and the likelihood that antibodies generated to

target metagenomic fragments will also target human

proteins (target "self") is ﬁnite.

A litany of research implies a re-evaluation of the

"autoantibody." Recently researchers have shown that cer-

tain autoantibodies are created in response to several

well-studied pathogens. "Lupus speciﬁc autoantibodies"

such as RO, La or dsDNA are often generated in response

to Epstein-Barr Virus.146

Similarly, anti-EBNA-1 antibodies

are able to bind lupus-speciﬁc autoantigens such as Sm or

Ro.Harley146 Casali and Slaughter found that in humans,

EBV is a polyclonal B cell activator, and in vitro transforma-

tion with EBV results in production of rheumatoid factor

(RF).147, 148 Possnett et al. argues that high titers of RF are

associated with severe rheumatoid arthritis but also ap-

pear in a number of other diseases including viral, bacte-

rial, and parasitic infections.149

Maturation of RF can be

initiated by chronic infections.150 For example, patients

with subacute bacterial endocarditis, which is frequently

tied to the presence of Streptococcus, also often present

with high levels of RF.151 Williams et al. showed that once

the oﬀending infectious agent is removed with antibiotic

therapy, the RF disappears.152 Similarly, the autoimmune

disease thrombocytopenic purpura (ITP) is mediated by

what are considered to be anti-platelet autoantibodies.

However, Asahi et al. found that eradication of H. pylori is

eﬀective in increasing platelet count in nearly half of ITP

patients infected with the bacterium.153 Barzilai and team

also found that Hepatitis B shares amino acid sequences

with diﬀerent autoantigens, further suggesting that so-

called autoantibodies may actually be created in response

to pathogens.146

Autoantibodies have been detected in

patients without autoimmune disease during periods of

infection. Berlin et al. collected sera from 88 patients with

acute infections (41 bacterial, 23 viral, 17 parasitic, and 7

rikettsial.154 Elevated titers of autoantibodies including

annexin-V, prothrombin, ASCA, ANA, or antiphospholipid

antibodies were detected in approximately half of the

subjects, with 34 individuals harboring elevated titers of

at least two "autoantibodies."

EBV, E. coli, Salmonella and other pathogens discussed

above are easily detected by culture-based methods that

may explain why their presence has already been tied to

"autoantibody" production. Yet the vast majority of the

human microbiota is understudied. This means that what

we now consider to be autoantibodies in many autoim-

mune diagnoses may also indicate the presence of patho-

gens, but pathogens that have yet to be fully character-

ized and named. Thus, in addition to looking for antibod-

ies to well-characterized pathogens, it is also important

that we look for antibodies indicating the presence of the

underlying chronic microbiota, some of which we may

also be mistaking for autoantibodies. Like the pathogens

that may create them, many of these antibodies may not

yet be detected by standard testing. If this is the case,

hundreds of pathogen-induced antibodies may exist and

impact the autoimmune disease state, but the possible

detection and correlation of such antibodies with speciﬁc

components of the microbiota remains diﬃcult until a

much larger portion of the microbiota has been character-

ized.

Because many antibodies demonstrate a high degree of

polyspeciﬁcity, it is possible that in some cases, antibodies

initially directed against pathogens could also attack hu-

man tissue.155 According to Bozic, oxidative alterations,

aﬀecting either the hypervariable region or the receptor

site of IgGs, may inﬂuence their functions.156

Similarly,

McIntyre reported the appearance and disappearance of

antiphospholipid antibodies subsequent to oxidation re-

actions in human blood.157 Dimitrov et al. has shown that

Proal et al.: Autoimmune disease and the human metagenome 21

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

a fraction of antibodies present in all healthy individuals

begin to recognize large number of self-antigens only af-

ter a transient exposure to certain protein-destabilizing

conditions, including low or high pH, high salt concentra-

tion, chaotropic factors and redox-active agents.158 This

points to at least one mechanism whereby the oxidative

stress that accumulates in inﬂamed tissue could be at

least partly responsible for the apparent polyspeciﬁcity of

antibodies and autoantibodies.

Molecular mimicry, in which peptides from pathogens

share sequence or structural similarities with self-

antigens, may also contribute to autoantibody produc-

tion. Lekakh et al. found that autoantibodies with poly-

speciﬁc activity in the serum of healthy donors were able

to cross-react with DNA and lipopolysaccharides (LPS) of

widespread species of bacteria including E. coli, P. aerugi-

nosa, Shigella boydii, and Salmonella.159 Crohn's disease is

classiﬁed as an autoimmune condition based largely on

the presence of perinuclear anti-nuclear cytoplasmic anti-

bodies (pANCA) in patients with the disease. Yet recently

two major species of proteins immunoreactive to pANCA

were detected in bacteria from anaerobic libraries, impli-

cating colonic bacteria as a possible trigger for the

disease-associated immune response.

We previously discussed how factors other than calorie

consumption may contribute to the weight gain often

associated with autoimmune or inﬂammatory conditions.

Fetissov et al. studied healthy women for the presence of

IgG or IgA autoantibodies directed against 14 key regula-

tory peptides and neuropeptides including ghrelin, leptin,

vasopressin, and insulin.108 They found numerous cases of

sequence homology among these peptides and the pro-

tein structures of over 30 microbes including Lactobacilli,

H. pylori, E. coli, Yersinia pseudotuberculosis, and Listeria

monocytogenes, suggesting that the "autoantibodies"

were actually the result of molecular mimicry. In the pres-

ence of certain pathogenic bacterial species, the produc-

tion of IgG autoantibodies directed against ghrelin were

upregulated, suggesting a complex interplay between

autoantibody levels and microbial antigens. This sug-

gested that these so-called "autoantibodies" might not

only have physiologic implications in pathways that regu-

late hunger and satiety but also represent a key link be-

tween the gut and the brain.

An increasing number of studies also show that what are

currently perceived as autoantibodies can often be de-

tected in so called healthy individuals years before the full

presentation of an autoimmune disease state. Many re-

searchers now espouse that early detection of these anti-

bodies can help predict whether or not such a "healthy"

person will develop an autoimmune disease. For example,

in an 8-year prospective study, Swaak et al. examined

the diagnostic signiﬁcance of anti-double-stranded de-

oxyribonucleic acid (anti-dsDNA) determination in a

group of 441 patients without systemic lupus erythema-

tosus whose sera were found to contain antibodies to

dsDNA on routine screening.160 Within one year, 69% (304)

of these patients fulﬁlled the preliminary American

Rheumatism Association (ARA) criteria for systemic lupus

erythematosus (SLE). Eighty-two of the remaining 137 pa-

tients were followed up for several years. At the end of the

study, 52% of these patients had also developed systemic

lupus erythematosus. The team concluded that about

85% of patients without systemic lupus erythematosus

with anti-dsDNA in the circulation would develop SLE

within a few years.

Another recent study of blood from 441 healthy Portu-

guese blood-donors found autoantibodies for rheumatoid

factor, anti cyclic citrunillated peptides, anti-mitochondria,

anti-Sacharomyces cerevisiae, ANA, anti-TTG, and anti-

Beta2- glycoprotein.161 More than 30% of the blood con-

tained one or more of the antibodies, 4% exhibited two

antibodies, and nearly 1% had three or more antibodies

present. It is clear that sub-clinical autoimmune disease is

much more common than previously thought.

This gradual presentation of an increasing number of so-

called "autoantibodies" in the years before a patient

meets the oﬃcial criteria for an autoimmune diagnosis

supports the model of successive infection described ear-

lier -- pathogenic components of the microbiota gradually

accumulate over the course of a lifetime until bacterial,

viral and phage load reaches a level at which a diagnosis

can be made. It also supports the contention that indi-

viduals perceived as "healthy" may still harbor and accu-

mulate pathogenic microbes that will eventually lead to

an inﬂammatory diagnosis, or a process associated with

"aging." Indeed, it is possible that any antibodies that

damage “self” do so as an unintended polyspeciﬁc conse-

Proal et al.: Autoimmune disease and the human metagenome 22

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

quence of their activity against the metagenomic patho-

gens.

Therapies in the era of the metagenome

At the 2008 International Conference on Metagenomics in

La Jolla, CA, James Kinross of the Imperial College of Lon-

don began his speech with the following statement: "We

surgeons have been operating on the gut for literally

thousands of years and the microbiota has just been this

extraordinary elephant in the room. We seem to have

completely ignored the fact that we've co-evolved with

thousands of bacteria over millions of years and that they

somehow may be important to our health. As doctors, we

routinely do terrible things to the microbiota and I'm sure

this has implications for our health."

While most physicians are undoubtedly well-intentioned,

Kinross is correct in that many clinicians are generally not

oﬀered training that would keep them up to date with

advances in metagenomics. The result is that many doc-

tors still believe that non-mucosal surfaces of the body are

largely sterile and that bacteria and other pathogens are

not driving factors in the autoimmune processes. Instead,

the standard of care for patients with autoimmune dis-

ease continues to be corticosteroids and TNF-alpha block-

ing medications. According to a 2008 report, TNF-alpha

inhibitors accounted for 80% of rheumatoid arthritis drug

sales in the United States, France, Germany, Italy, Spain,

the United Kingdom and Japan. Use of these immuno-

suppressants is still grounded in the theory that autoim-

mune disease results from an overly exuberant immune

response, and these drugs are administered without con-

sideration for the presence of a metagenome. Whether

helpful or harmful, there is no question that by dramati-

cally slowing the immune response, such therapies must

necessarily and profoundly aﬀect the composition, devel-

opment, and stability of the human microbiota.

Despite the copious use of these immunosuppressant

drugs in autoimmune conditions, they provide, at best,

short-term palliation. Gottlieb et al. showed that steroid

use causes relapse in sarcoidosis.162 Additionally, there are

no deﬁnitive studies showing corticosteroids improve

long-term prognosis in the treatment of chronic inﬂam-

matory illness, nor is there any demonstrated reduction in

mortality. Van den Bosch and Grutters write, “Remarkably,

despite over 50 years of use, there is no proof of long-term

(survival) beneﬁt from corticosteroid treatment.”163 On the

other hand, one of the side eﬀects of TNF-alpha inhibitors

is an increased risk of tuberculosis. Several studies have

show that TNF-alpha production is required for the proper

expression of acquired speciﬁc resistance following infec-

tion with M. tuberculosis.164, 165 So if we inhibit TNF-alpha

expression, we would expect a long-term increase in the

prevalence of not only tuberculosis, but in any of the

autoimmune or inﬂammatory diseases already associated

with chronic forms of mycobacteria and other bacteria.166,

167

The failure of these ﬁrst-line therapies to cure "autoim-

munity," and the range of detrimental side eﬀects associ-

ated with their use, suggests that slowing the immune

response of patients with autoimmune disease is coun-

terproductive, allowing microbial populations to develop

unchecked. Now that autoimmune conditions are more

widely understood as illnesses in which myriad pathogens

may trigger or drive the disease process, eﬀorts to target

the root cause of autoimmune disease should instead be

targeted towards activating the innate immune response,

not suppressing it.

Our own work100 oﬀers an example of the results of stimu-

lating rather than suppressing the innate immune re-

sponse of patients with autoimmune disease. Over the

past seven years, we have observed the eﬀects of an ex-

perimental therapy for autoimmune disease that uses the

VDR agonist olmesartan to reverse pathogen-induced

VDR dysregulation. Subjects are also administered subin-

hibitory bacteriostatic antibiotics, which weaken bacterial

ribosomes so that pathogens can more easily be targeted

by the reactivated immune system. Nearly all of the hun-

dreds of patients to start the therapy reported the pre-

dicted increase in speciﬁc symptoms of their autoimmune

diagnosis. After months, or sometimes years, of dealing

with these symptomatic ﬂares, the very symptoms that

waxed and waned in synchronism with antibiotic admini-

stration began to disappear, resulting in improvement

and, in many cases, eventual resolution of the disease

process. This response has been noted in the widely vary-

ing diagnoses sarcoidosis, rheumatoid arthritis, lupus,

type II diabetes, uveitis, Hashimoto's thyroiditis, ankylos-

ing spondylitis, chronic fatigue syndrome, and ﬁbromyal-

gia among others. The often dramatic elevations in dis-

Proal et al.: Autoimmune disease and the human metagenome 23

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

ease activity observed among study subjects - particularly

during the early stages of therapy - cannot be attributed

to side eﬀects of the protocol medications, as individually

the drugs are well known and unremarkable.168

Addition-

ally, when healthy individuals have been administered the

same medications they do not suﬀer any similar symp-

toms.

The most viable hypothesis for these temporary surges in

disease symptoms and inﬂammatory markers is that

treatment medications allow the immune system to

mount an eﬀective attack on an intracellular microbiota,

such as the microbiota observed by Wirostko et al. It is

reasonable to expect that when intraphagocytic patho-

gens are killed, that some of the host cells will also un-

dergo apoptosis, phagocytosis or simply disintegration,

leading to an increase in inﬂammation. For over 100 years,

researchers have noted that the death of acute and persis-

tent pathogens is accompanied by a surge in inﬂamma-

tion. They have attributed the temporary rise in inﬂamma-

tion to an increase in endotoxin and cytokine release

upon bacterial death. Known as the Jarisch-Herxheimer

Reaction, or immunopathology, this phenomenon has

been previously demonstrated after antibiotic administra-

tion in diseases including tuberculosis,169

borreliosis,170

tick-borne relapsing fever,171 multiple sclerosis,172 Whipple

disease,173

and syphilitic alopecia

174 among others.

Martin Zinkernagel also observed immunopathology in

the mice he had infected with a persistent neuro-active

virus.175 Similarly, immune reconstitution inﬂammatory

syndrome (IRIS) is a condition seen in some cases of AIDS

following the use of antiretroviral drugs. As the immune

system begins to recover, it responds to previously ac-

quired opportunistic infections with an overwhelming

inﬂammatory response that, like the immunopathological

reaction we observe, makes the symptoms of the infec-

tion temporarily worse.176 At this point in time, the exact

species or forms of bacteria potentially killed by any one

subject in our own study cohort remain unknown. As the

focus of the Human Microbiome Project moves beyond

the mucosal surfaces, and catalogs L-forms and other in-

tracellular species within body tissues, a clearer picture of

disease pathogenesis will emerge. However, as long as

patients continue to report improvement and recovery,

determining the exact nature of pathogens being tar-

geted by the therapy has not been a high priority, given

the limited resources currently allocated to this research

team.

Some subjects in the cohort have reported drops in viral

titers, suggesting that once the immune system is no

longer burdened by the pathogenic components of the

bacterial microbiota, it may regain the ability to target

chronic viruses as well. This suggests that treatments that

reverse immunosuppression caused by the bacterial mi-

crobiota might also prove useful in mitigating viral viru-

lence.

Our research suggests that while some people report be-

ing "allergic" to certain bacteriostatic antibiotics, what

they perceive as an "allergy" may actually be immunopa-

thological reactions. For example, there are reports of

minocycline "inducing lupus."177 A more logical explana-

tion may be that certain patients harbor persistent bacte-

rial species that predispose for sub-clinical lupus. When

minocycline is administered, some of these bacteria are

killed, resulting in immunopathological reactions that are

mistakenly interpreted as clinical manifestation of the dis-

ease.

What we have initiated needs further testing. However,

the reports of profound immunopathological reactions in

autoimmune subjects imply the need to re-examine

whether palliative drugs actually provide long-term

beneﬁt for patients with autoimmune disease. Whether at

the doctor's oﬃce or the health food store, patients with

autoimmune conditions continually seek out palliative

drugs or supplements that successfully reduce symptoms

by lowering inﬂammation. Yet, if bacteria drive the patho-

genesis of autoimmune inﬂammation, and chronic bacte-

rial death invariably results in temporary increases in dis-

comfort, then treatments that mitigate symptoms may

well do so at the expense of proliferation in pathogenic

components of the microbiota. Commonly used immuno-

suppressive compounds include vitamin D which, al-

though its immunosuppressive properties have now been

identiﬁed,178 is now viewed as the ultimate inexpensive

wonder drug.179

Frequent use of vitamin D, as well as

other substances that slow immune activity, could at least

partially account for the recently increased prevalence of

nearly every autoimmune disease.180

Proal et al.: Autoimmune disease and the human metagenome 24

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

L-form bacteria: an often overlooked compo-

nent of the microbiota

Certain stages of the bacterial life cycle result in the loss of

the cell wall. L-form bacteria are often less than 0.2 µm in

diameter,8 and are therefore diﬃcult to view with a stan-

dard optical microscope. Not only do these L-form vari-

ants fail to succumb to antibiotics that target the bacterial

cell wall, those antibiotics encourage the formation of L-

forms. “Treatment with penicillin does not merely select

for L-forms (which are penicillin-resistant) but actually in-

duces L-form growth,” states Josep Casadesus of the Uni-

versity of Sevilla.181 In fact, researchers deliberately culture

classical forms of bacteria in conjunction with various

beta-lactam antibiotics in order to create L-forms.1 The

ability of the L-form to ﬂourish in the face of treatment

with the beta-lactam antibiotics points to a mechanism by

which acute bacterial forms can mutate into latent mu-

tants that may cause disease at a later time. Some re-

searchers have deemed the conversion into the L-form

state to be a universal property of bacteria.182

Joseleau-Petit et al. showed that classical forms of bacteria

transform into the L-form only if they are denied the abil-

ity to form a normal cell wall.183 The beta-lactam antibiot-

ics work towards this end by blocking the creation of

penicillin-binding proteins (PBPs) – proteins responsible

for forming the cross-linked chains associated with a

peptidoglycan-derived cell wall. When the ability of the

PBPs to create a full cell wall is blocked, the cells also be-

come spherical and osmosensitive. Recently, Glover et al.

performed the ﬁrst systematic genetic evaluation of genes

and pathways involved in the formation and survival of

unstable L-form bacteria.184 Microarray analysis of L-form

versus classical bacterial colonies revealed many up-

regulated genes of unknown function as well as multiple

over-expressed stress pathways shared in common with

persister cells and bioﬁlms. Dell'Era et al. also observed

cell division and changes in gene expression in stable L.

monocytogenes L-forms.185

Since the discovery of the L-forms in 1935,186 they have

been described in hundreds of publications. Yet because

researchers are only just beginning to use molecular tools

to study the L-form, they are still seldom factored into the

mix of microbes that compose the human microbiome.

However, over the years, L-forms have been implicated in

dozens of diseases of unknown etiology including rheu-

matoid arthritis, multiple sclerosis, sarcoidosis, glomeru-

lonephritis, idiopathic hematuria, interstitial cystitis,

rheumatic fever, and syphilis – as well as a large number

of chronic and relapsing infections.1, 8

A research consideration: men are not tall mice

without tails

The emerging role of the human microbiota implies a re-

consideration of certain longstanding and frequently in-

voked models of disease. According to Javier Mestas of

University of California, Irvine, “There has been a tendency

to ignore diﬀerences and in many cases, perhaps, make

the assumption that what is true in mice is necessarily

true in humans. By making such assumptions we run the

risk of overlooking aspects of human immunology that do

not occur, or cannot be modeled, in mice.”187 Murine

models are still used in an eﬀort to understand most auto-

immune and inﬂammatory conditions, despite the obvi-

ous diﬀerences between the murine and human immune

systems.

For example, there are major diﬀerences in the Toll-Like

Receptors. TLR1-9 exist in both mouse and man, although

TLR8 detects single stranded RNA in man and has no

known function in the mouse. TLR10 exists in humans

only; it is a degenerative pseudo-gene in the mouse.

TLR11,12 and 13 in mice do not exist in man and their

function is not yet well deﬁned.

Analysis of the human and murine VDR oﬀers other ex-

amples of discord between man and mouse. Marshall's

molecular dynamics emulation showed that the drug

olmesartan, a putative VDR agonist, binds into a diﬀerent

conformation in the murine VDR to that of Homo sapi-

ens,188 calling into question the whole concept of drug

safety testing in murine models.

While the human VDR transcribes dozens of genes neces-

sary for a robust innate immune response, including many

key antimicrobial peptides, the Vitamin D Receptor does

not similarly control the murine innate immune system.

The murine innate immune response is dependent on a

cascade of nitric oxide functions in a manner yet to be

fully understood.189 Although mice have VDRs, the ho-

mology diﬀers, and they express diﬀerent genes than the

Proal et al.: Autoimmune disease and the human metagenome 25

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

human VDR. For example, the gene encoding the calcium

binding protein osteocalcin is “robustly” transcribed by

the VDR in humans, but not in mice.

Brahmachary et al. showed that the rat VDR does not ex-

press the cathelicidin antimicrobial peptides (AMPs),

marking an important diﬀerence in the way the two spe-

cies target invading pathogens.72

Gombart et al. recently

expanded on the ﬁnding by providing evidence of an evo-

lutionarily ﬁxed, Alu-mediated divergence in steroid hor-

mone nuclear receptor gene regulation between humans/

primates and other mammals.190 This divergence, which

placed the cathelicidin pathway under VDR control only in

humans and closely related primates, remained under pu-

rifying selection for the last 55-60 million years, and yet

even cathelicidin in primates is not identical to that in

man. Eventually, the pathway evolved to become a key

component of a novel innate immune response unique to

human infection. Because the murine VDR does not ex-

press cathelicidin, there is less of an evolutionary incen-

tive for components of the murine microbiota to dysregu-

late its expression. This suggests that the survival mecha-

nisms employed by the human and murine microbiotas

may be very diﬀerent. Thus, the intermingling of murine

and human biologies in the literature hinders our ability

to fully understand nuclear receptor control of the AMPs

and other key aspects of innate immunity.

Discussion

The prevailing theory of autoimmune disease, which dic-

tates that the body creates autoantibodies that attack its

own cells, was developed during an era when culture-

based methods vastly underestimated the number of mi-

crobes capable of persisting in and on Homo sapiens. The

advent of culture-independent tools such as 16S RNA se-

quencing, single cell sampling, and pyrosequencing have

opened the door to an era of discovery. Rather than a ster-

ile compartment, the human body is now known to teem

with thousands of species of bacteria, viruses and phages.

In addition to persisting on the body's external surfaces,

these microbes survive in the blood and in many of the

tissues which become inﬂamed during autoimmune dis-

ease, suggesting that what were once thought to be

"autoimmune" processes may instead result from the

presence of persistent microbes. Metagenomics is allow-

ing us to study these microbes in the tissues within which

they naturally persist, where they can be examined in the

context of other microbes in their community. A more ex-

act understanding of how networks of microbes can in-

teract to cause disease has superseded Koch’s Postulates,

which stipulate that a single microbe causes a single dis-

ease.

While diseases were once categorized largely on the basis

of symptom presentation, they can now be classiﬁed

based on their underlying genetics. Yet the expression

of key human genes is continually altered by a plethora of

microbial metabolites through an almost imponderable

number of interactions. These metabolites, some of which

are created by bacteria considered to be "friendly" or in-

nocuous, can directly drive the pathogenesis of autoim-

mune disease by altering the expression of genes such as

ACE and PTN22, genes associated with diagnoses includ-

ing rheumatoid arthritis, lupus, diabetes mellitus, myocar-

dial infarction, renal tubular dysgenesis and Alzheimer's. It

is becoming apparent that autoimmune processes cannot

be fully understood if the human genome is studied in

isolation. An understanding of the interactions between

the human genome and the metagenome calls for a more

nuanced understanding of the microbiota. Classifying cer-

tain microbes as purely commensal may underrepresent

the full spectrum of their actions. Indeed, harmless spe-

cies of bacteria and viruses can easily acquire virulent

plasmids via horizontal gene transfer or homologous re-

combination.

The microbiota has persisted in and on the human body

for millennia. It has evolved to slow the host immune re-

sponse in order to ensure microbial survival. Pathogens

such as M. tuberculosis, Borrelia, Epstein-Barr virus, and HIV

have evolved to dysregulate the VDR nuclear receptor, in-

hibiting expression of the beta-Defensin and cathelicidin

antimicrobial peptides along with TLR2. Flow-on eﬀects

from VDR dysregulation can further alter AMP expression

via (at least) the alpha-thyroid, androgen and glucocorti-

coid nuclear receptors. This may result in the immuno-

suppression and hormonal imbalances characteristic of

many autoimmune diagnoses.

The bacteria that cause autoimmune disease likely accu-

mulate over a lifetime, with individuals picking up patho-

gens with greater ease over time, as the immune response

Proal et al.: Autoimmune disease and the human metagenome 26

This is a preprint of a book chapter published by Springer Media, (c) Copyright, 2011, Springer Media

becomes increasingly constrained. Successive infection

dictates that even people with the same autoimmune di-

agnosis are unlikely to present with identical clusters of

symptoms and helps explain the high levels of comorbid-

ity observed among these patients. Common autoim-

mune comorbidities include inﬂammatory conditions

such as cardiovascular disease, along with mental diagno-

ses such as depression or anxiety, suggesting these condi-

tions may also be driven by the microbiota. Thus, insights

gained from studying microbial composition in autoim-

mune disease can accelerate research in other areas of

medicine. Recently, several studies have shown the pres-

ence of "autoantibodies" in autism with anti-nuclear anti-

body seropositivity showing a signiﬁcant positive associa-

tion with disease severity, mental retardation and electro-

encephalogram abnormalities. Rather than assign autism

to the end of a growing list of autoimmune diagnoses, this

knowledge might be better used as a basis on which to

further explore the role that components of the microbi-

ota may play in driving the pathogenesis of disease.

Analyzing autoimmune disease through the lens of meta-

genomics calls for a re-evaluation of the autoantibody.

Polyspeciﬁc autoantibodies are increasingly being associ-

ated with elements of the microbiota, making it likely that

the term "autoimmune" will soon lose its diagnostic utility.

When a disabled immune system is forced to respond to

the presence of a chronic microbiota, the resulting cas-

cade of cytokines and chemokines will stimulate an adap-

tive immune response. The adaptive immune system will

then proceed to generate antibodies to fragments of DNA

generated by apoptosis or phagocytosis of infected cells.

This is supported by studies showing that so-called auto-

antibodies such as RO, La, dsDNA and RF can be created in

response to various bacterial and viral pathogens. Autoan-

tibodies are often observed before a patient becomes

fully symptomatic with an autoimmune diagnosis, reﬂect-

ing the gradual accumulation of persistent microbes.

Rather than focusing on phenotypes and subsets of the

metagenome, microbiome research may instead beneﬁt

from broader approaches geared toward understanding

shared mechanisms of persistence. Translational medicine

should aim at cutting through barriers among specialties,

even between biologists and clinicians, so that more of

the pieces of the emerging jigsaw of disease etiology can

drop into place, and autoimmune disease patients can

fully beneﬁt from the insights gained from metagenomic

science.

Acknowledgements

The authors wish to acknowledge the assistance of Dr.

Elena Kashuba for sharing her data and helping us pre-

pare Figure 2.

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Proal et al.: Autoimmune disease and the human metagenome 33

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Autoimmune Disease and the Human Metagenome

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