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Autoimmune Disease and the Human Metagenome

  • PolyBio Research Foundation
  • Autoimmunity Research Foundation

Abstract and Figures

The prevailing theory of autoimmune disease, that the body creates autoantibodies that attack “self,” was developed during an era when culture-based methods vastly underestimated the number of microbes capable of persisting 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 vitamin D receptor nuclear receptor, affecting the expression of endogenous 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 microbiota. This helps explain the high levels of comorbidity observed among patients with autoimmune conditions. What are commonly believed to be autoantibodies may instead be created in response to this metagenomic microbiota when the adaptive immune system is forced to deal with disintegration of infected cells. Similarly, haplotypes associated with autoimmune conditions vary widely among individuals and populations. They are more suggestive of a regional infectious model rather than a model in which an illness is caused by inherited variation of HLA haplotypes
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Life Sciences - Ecology | Metagenomics of the Human Body
© Springer. Part of Springer Science+Business Media
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
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
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
1 of 1 8/17/2010 7:55 A
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
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, aecting 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 dierent 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
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
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 ecacy 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
eorts to alter the pH and growth medium of their sam-
ples in an eort 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 oered 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
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 ocials, 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 eec-
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 dierent 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 eort 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 diers 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 dierences 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 dierent 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. Dierent 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-
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 dierent
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
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
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 eective 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 aect 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 aect 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
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
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
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
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 eorts to understand how
these proteins aect 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
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 dierent 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 dierent. 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 dierences 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 dierent 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 dierent 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
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
o 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-
Proal et al.: Autoimmune disease and the human metagenome 8
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rate bacterial species, Homaster 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
Homaster 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
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 aect 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-
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
“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 eector 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 oers 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 anity
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 anity for the thyroid beta, suggesting that it
can displace T3 and T4 from the binding pocket (Table
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 aected.
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 eects that potentially
disable the bulk of the body's antimicrobial peptides. A
patient aected 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
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-
Proal et al.: Autoimmune disease and the human metagenome 12
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Table 1. Affinities of Native Ligands and 1,25-D for Various
Nuclear Receptors
Nuclear receptor
ligand (Kd)
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 aected 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 suer
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
eect. This process is known as successive infection and
oers 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
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 dierent
times. The immune response could be expected to be par-
Proal et al.: Autoimmune disease and the human metagenome 13
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ticularly weak after traumatic events such as surgery, a car
accident or even a pregnancy.80
People accumulate microbiota-altering pathogens in myr-
iad dierent 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
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 dierent
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 suers 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 dierent 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 eects 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 suered 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
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 suer 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
Thus the catastrophic failure of the human metabolism
we see in autoimmune disease which at first glance ap-
pears so diverse and so dierent among dierent 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 diers
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
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patients suering from one inflammatory disease and the
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 suer 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 suer
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
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 eect
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 eective 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-
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 dicult
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
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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 dierent. 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
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 dierent 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.
Homan 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 dicult 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
For example, in cancer, for nearly all regions
conclusively identified by genome wide association stud-
ies (GWAS), the per allele eect sizes estimated are less
than 1.3. While over 85 regions have been conclusively
associated in over a dozen dierent 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 dier 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 eect 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
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 eort 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 eectively eradicate
these conditions.
It may be too early to call human genomic research an
unqualified failure,131
but it is dicult 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
oers a survival advantage. For example, the genetic dis-
orders cystic brosis 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
dierent in aortic tissue than in blood samples from the
same patients,140
Gottlieb remarked, "Genome-wide asso-
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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 dierent genetic sequences could arise through
selective pressure in dierent 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 diers 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-
Proal et al.: Autoimmune disease and the human metagenome 20
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pectedly match a probe, and increases the possibility of a
false-positive being signaled for the particular SNP being
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 oending 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
eective 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 dierent 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 dicult until a
much larger portion of the microbiota has been character-
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,
aecting either the hypervariable region or the receptor
site of IgGs, may influence their functions.156
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
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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 ocial 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
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quence of their activity against the metagenomic patho-
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
oered 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 aect 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 eects 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,
The failure of these first-line therapies to cure "autoim-
munity," and the range of detrimental side eects 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, eorts to target
the root cause of autoimmune disease should instead be
targeted towards activating the innate immune response,
not suppressing it.
Our own work100 oers 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 eects 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 eects of the protocol medications, as individually
the drugs are well known and unremarkable.168
ally, when healthy individuals have been administered the
same medications they do not suer any similar symp-
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 eective 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
tick-borne relapsing fever,171 multiple sclerosis,172 Whipple
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
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-
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-
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 oce 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 dicult 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 dierences 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 eort to understand most auto-
immune and inflammatory conditions, despite the obvi-
ous dierences between the murine and human immune
For example, there are major dierences 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 oers 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 dierent
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 diers, and they express dierent 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 dierence 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 dierent. 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.
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-
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-
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 eects
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
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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... Several studies have demonstrated that HDP expression regulation by VDR is not evolutionarily conserved, because it is restricted to humans and nonhuman primates and CAMP genes are not responsive to VDR in other animals, such as mice [28,36,38,39]. Thus, there is a difference between the immune responses of mice and humans [51]. Given that VDR and its calcitriol linker have a key role in the human immune response, current knowledge of the mechanisms involved in the HDP expression, especially those associated with nuclear receptors, and probably other aspects of innate immunity, is limited because mice are currently the most-studied animal model of the immune response [51]. ...
... Thus, there is a difference between the immune responses of mice and humans [51]. Given that VDR and its calcitriol linker have a key role in the human immune response, current knowledge of the mechanisms involved in the HDP expression, especially those associated with nuclear receptors, and probably other aspects of innate immunity, is limited because mice are currently the most-studied animal model of the immune response [51]. ...