Autoimmunity Reviews 19 (2020) 102430

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Autoimmunity Reviews
journal homepage: www.elsevier.com/locate/autrev

Multiple sclerosis, the microbiome, TLR2, and the hygiene hypothesis T

a b a,c,⁎
Nicholas J. Wasko , Frank Nichols , Robert B. Clark
a
Department of Immunology, The University of Connecticut Health Center, Farmington, CT 06032, USA
b
School of Dental Medicine, Department of Oral Health and Diagnostic Sciences, The University of Connecticut Health Center, Farmington, CT 06032, USA
c
Department of Medicine,The University of Connecticut Health Center, Farmington, CT 06032, USA

A R T I C LE I N FO A B S T R A C T

Keywords: The pathophysiology of autoimmune diseases such as Multiple Sclerosis (MS) involves a complex interaction
Multiple sclerosis between genetic and environmental factors. Studies of monozygotic twins suggest a significant role for en-
Microbiome vironmental factors in susceptibility to MS. Numerous studies, driven by the “Hygiene Hypothesis,” have focused
TLR2 on the role of environmental factors in allergic and autoimmune diseases. The hygiene hypothesis postulates that
TLR tolerance
individuals living in environments that are too “clean” lack the requisite exposure to “immune-tolerizing” mi-
Remyelination
Hygiene hypothesis
crobial products, resulting in poorly regulated immune systems and increased immune-mediated diseases.
Interestingly, few studies have linked MS with the hygiene hypothesis. Similarly, although numerous studies
have examined the role of the microbiome in autoimmune diseases, there has been no consistent documentation
of disease-specific alterations in the MS microbiome. In this review, we present evidence that integrating the
hygiene hypothesis and the microbiome allows for the identification of novel pathophysiologic mechanisms in
MS.
Our central hypothesis is that the microbiome in MS represents a “defective environment” that fails to provide
normal levels of “TLR2-tolerizing” bacterial products to the systemic immune system. Consistent with the hy-
giene hypothesis, we posit that this defective microbiome function results in abnormally regulated systemic
innate immune TLR2 responses that play a critical role in both the inflammatory and defective remyelinative
aspects of MS. We have completed proof of concept studies that support the inflammatory, remyelinating, and
human immune response components of this paradigm. Our studies suggest that induction of TLR2 tolerance
may represent a novel approach to treating MS, inhibiting autoimmune inflammation while simultaneously
facilitating remyelination.

1. Introduction variables that contribute to pathogenesis, disease onset, and therapeutic

response. Genetic factors lay the foundation for autoimmune diseases,
The study of autoimmune diseases is complicated by the multiple but studies such as those in monozygotic twins demonstrate the

Abbreviations: APC, Antigen presenting cell; Arg1, Arginase 1.; CNS, Central nervous system.; DAMP, Damage associated molecular pattern.; EAE, Experimental
autoimmune encephalomyelitis.; ELISA, Enzyme-linked immunosorbent assay.; EM, Electron microscopy.; GI, Gastrointestinal.; IBA1, Ionized calcium binding
adaptor molecule 1.; iNOS, Inducible nitric oxide synthase.; IRAK, Interleukin-1 receptor-associated kinase.; i.v., Intravenous.; L654, Lipid 654.; LPS,
Lipopolysaccharide.; MRM-mass spectrometry, Multiple reaction monitoring mass spectrometry.; MS, Multiple sclerosis.; MyD88, Myeloid differentiation primary
response protein MyD88.; NF-κB, nuClear factor kappa-light-chain-enhancer of activated B cells.; OL, Oligodendrocyte.; OPC, Oligodendrocyte precursor cell.; P2C,
Pam2CSK4, a synthetic diacylated lipopeptide.; P3C, Pam3CSK4, a synthetic triacylated lipopeptide.; PAMP, Pathogen associated molecular pattern.; PBMC,
Peripheral blood mononuclear cells.; PLP, Proteolipid protein.; PPMS, Primary progressive multiple sclerosis.; PRR, Pattern recognition receptor.; RRMS, Relapsing
remitting multiple sclerosis.; SPMS, Secondary progressive multiple sclerosis.; TID, Type 1 diabetes.; TIR, Toll/interleukin-1 receptor (TIR) homology domain.; TLR,
Toll-like receptor.; Traf6, TNF Receptor-associated factor 6.; Treg, Regulatory T cell.; TRIF, TIR-domain-containing adapter-inducing interferon-β.; TNFα, Tumor
necrosis alpha.; VC, Vehicle control.; Animal welfare, All mice were maintained under specific pathogen-free conditions in accordance with the guidelines for the
Center for Comparative Medicine at UConn Health. All procedures were performed in compliance with Institutional Animal Care and Use Committee-approved
protocols.; Informed consent, Informed consent was obtained on all human subjects after the nature and possible consequences of the studies had been fully
explained.
⁎
Corresponding author at: Department of Immunology, Department of Medicine, Room E3013, University of Connecticut Health Center, 263 Farmington Avenue,
Farmington, CT 06030, USA.
E-mail addresses: wasko@uchc.edu (N.J. Wasko), nichols@uchc.edu (F. Nichols), rclark@uchc.edu (R.B. Clark).

https://doi.org/10.1016/j.autrev.2019.102430
Received 7 July 2019; Accepted 11 July 2019
Available online 15 November 2019
1568-9972/ © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
N.J. Wasko, et al.
significance of non-genetic factors in the etiology of these diseases.
Studies of monozygotic twins document the variable heritability for
different autoimmune diseases, ranging from high concordance among
twins with psoriasis to lower concordance (approximately 30%) among
twins with multiple sclerosis (MS) [1,2]. The twin concordance studies
in MS suggest that genetics can only partly explain the susceptibility for
developing MS and that environmental factors contribute significantly
to disease occurrence. In this way, environmental factors also take on
great significance as potential targets for new therapeutic approaches.
However, the “environment” involves many potentially relevant and
varied components such as prior infections, geography, toxin exposures
(including smoking), and diet, but discerning the relative importance of
such factors has proven difficult. Recently, much attention has been
focused on environmental factors and their role in allergic and auto-
immune diseases based on two major paradigms: 1) the “Hygiene Hy-
pothesis” and; 2) the role of the microbiome. However, these two
concepts have rarely been integrated as a lens for understanding these
immune-mediated diseases. In this review, we present evidence for
using this integrated approach to identify novel pathophysiologic me-
chanisms underlying MS.
2. The hygiene hypothesis
One clue to the identity of relevant environmental factors may be
found in the observation that the frequency of both autoimmune dis-
eases and allergic diseases (including asthma) have been significantly
increasing in industrialized nations over the past few decades. This has
led to the “Hygiene Hypothesis,” which postulates that the increase in
these immune-related allergic and autoimmune diseases is a result of an
environment that is too clean and fails to appropriately stimulate,
educate, and thus regulate the immune system [3]. The “cleanliness” of
the environment in most cases refers to the relative paucity of microbes
and microbial-derived molecules (pathogen-associated molecular pat-
terns or “PAMPs”) that are routinely encountered in one's normal daily
activities. Although the mechanisms underlying the hygiene hypothesis
have been somewhat elusive, recent studies have suggested that PAMP-
exposure that is chronically “too low” results in poorly regulated and
thus potentially over-reactive innate immune responses [4–6].
As highlighted by Stein et al. [5], a number of epidemiologic studies
conducted in Europe have shown significant protection from asthma
and allergic disease in children raised on traditional dairy farms with
high microbial and farm animal exposures [7–10]. In attempting to
identify relevant mechanisms underlying this association, Schuijs et al.
reported that chronic exposure to low dose lipopolysaccharide (“LPS”;
“endotoxin”) or farm dust extract protected mice from house dust mite-
induced asthma [4]. After toll-like receptor (TLR4) signaling, nuclear
translocation of NF-kB induces both pro-inflammatory genes and genes
for molecules such as A20 that attenuate signaling [4]. Schuijs et al.
generated mice that lacked A20 in lung epithelium and found that
treatment of mice with low dose LPS or farm dust extract no longer
suppressed asthma in these mice. These studies suggested that treat-
ment with low dose LPS or farm dust extract mediated suppression from
asthma through A20 and attenuation of TLR4 signaling. Consistent with
their mouse results, these investigators documented that a single nu-
cleotide polymorphism in the gene encoding A20 was associated with
allergy and asthma risk in children growing up on farms [4].
Stein et al. investigated the incidence of asthma in farming popu-
lations that are genetically similar but differ in their farming techniques
such that exposure to environmental PAMPs (specifically LPS in farm
dust) differ significantly between the populations [5]. These in-
vestigators found that the population with chronically high PAMP ex-
posures (i.e., “dirtier” environments) had a significantly lower in-
cidence of asthma compared to the population with the chronically low
PAMP exposures (i.e., “cleaner” environments). They concluded that
the more microbial products in the environment the less asthma in the
population, and they next went on to explore potential mechanisms
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Autoimmunity Reviews 19 (2020) 102430
underlying these findings [5]. They found that the level of environ-
mental PAMPs directly affected the phenotype and responses of cells of
the innate immune system. They documented that the population with
less PAMP exposure (i.e., the “cleaner” environment with more asthma)
demonstrated stronger innate immune responses (e.g., higher LPS-sti-
mulated cytokine secretion), while the population with more PAMP
exposure (i.e., the “dirtier” environment with less asthma) demon-
strated weaker innate immune responses and also demonstrated a more
“suppressed” peripheral blood monocyte phenotype (e.g., lower ex-
pression of HLA-DR molecules) [5]. The studies of Schuijs et al. [4] and
Stein et al. [5] highlight that the level of exposure to environmental
PAMPs plays a role in regulating the level of innate immune responses
in the host, and in this way might be a critical factor, along with ge-
netics, in determining the incidence of immune-mediated diseases.
3. Multiple sclerosis and the hygiene hypothesis
MS is a chronic neuroinflammatory disease affecting 2.3 million
people worldwide [11] characterized by immune-mediated damage to
the myelin sheath surrounding neuronal axons [12]. Roughly 80% of
cases present as relapsing-remitting MS (RRMS), with periodic clinical
attacks punctuating periods of stability. Many such cases transition into
secondary progressive MS (SPMS), in which neurological disability
accumulates more consistently [12]. Alternatively, primary progressive
MS (PPMS) occurs in 15% of cases, manifesting as continuous dete-
rioration of neurological function [13]. The disease pathogenesis in
RRMS is believed to involve a T lymphocyte-directed attack on myelin
or oligodendrocytes, the myelin producing cells, resulting in areas of
demyelination in the central nervous system (CNS). In addition, pa-
tients with MS have a defect in their ability to repair (remyelinate) their
demyelinated lesions. Consistent with this concept of T cell-directed
damage, therapeutic approaches that target the immune system can
reduce symptomatic flares in RRMS. In contrast, the pathogenesis of
both SPMS and PPMS is less clear and no existing therapies demonstrate
consistent efficacy in either slowing the progression of the disease or in
repairing the myelin damage [14–16]. However, as reviewed by
Fraussen et al., the identification of meningeal ectopic B cell follicles in
SPMS and the successful use of B cell-depleting therapy in PPMS have
underlined the importance of B cells in progressive MS” [17].
While extensive research has been conducted over decades at-
tempting to identify epidemiologic and environmental factors that in-
fluence the incidence of MS, very little has been conclusively docu-
mented. The most frequently cited MS-relevant environmental factors
include: the incidence of MS increases as one moves farther away from
the equator; the incidence of MS increases with a chronic decrease in
exposure to sunlight; and the incidence of MS increases in those with a
low level of serum vitamin D [18]. Other environmental/epidemiologic
factors that have historically been implicated in susceptibility to MS
include a history of early contraction or severe infection with Epstein
Barr virus [18]. Importantly, little has been published connecting MS to
the hygiene hypothesis.
As summarized by Wendel-Haga and Celius [19] and by Correale
and Gaitán [20], for many years the risk of MS has been suggested to be
higher in individuals with high hygienic standards during childhood,
but relevant specific factors have not been identified. Conflicting results
have been reported regarding number of siblings, attendance in a day
care center, and exposure to animals during childhood in relation to MS
risk, but common childhood infections and vaccinations do not seem to
influence the risk of MS. In line with the hygiene hypothesis, two large
meta-analyses have recently shown that infection with Helicobacter
pylori is negatively correlated with MS [21,22].
4. The microbiome
The microbiome, a term used to describe the trillions of commensal
microorganisms that occupy niches on or within the human body, has
N.J. Wasko, et al. Autoimmunity Reviews 19 (2020) 102430

become an area of intense research focus because of its potential as a [37,38] and thus a deficiency in these bacteria and these fatty acids
regulator or modifier of human health, disease, and therapeutic re- could play a role in a decreased functional suppressive capacity of
sponsiveness. Microbiome species colonize several body sites ranging regulatory T cells in RRMS patients [36].
from the oral cavity to the skin to the vagina, but the largest population Overall, there is as yet little consensus in identifying MS-specific
of microorganisms reside within the gastrointestinal (GI) tract [23–28]. microbiome profiles. As summarized by Tremlett et al., most studies
Studies assessing the role of the microbiome in human health and pa- report subtle, rather than large, differences in the MS gut microbiota
thology are ongoing, though preliminary studies suggest that disturbing composition, and it is likely that further longitudinal studies of the
a “normal” microbiome can lead to disease-related alterations in im- microbiome in MS will be necessary to understand the influences of
mune function and metabolism. The composition of the gut microbiome ongoing inflammation, concomitant medications, and issues such as
has been shown to modulate immune cell activity in the host [3], with whether any microbiome changes pre-date or are a consequence of MS
segmented filamentous bacteria inducing intestinal Th17 production or its treatment [39].
[29] and strains of Clostridium and other short chain fatty acid-produ-
cing bacteria facilitating the accumulation of regulatory T cells (Tregs) 6. TLR2 signaling connects MS to both the microbiome and the
in the gut [30]. Important questions that remain unanswered in mi- hygiene hypothesis
crobiome research include the identification of what constitutes a
“normal” microbiome [23,25] and the identification of mechanisms Over the past decade our laboratory has investigated whether MS
that underlie the effect of the microbiome on both the local and sys- could have any connection to the hygiene hypothesis and/or to the
temic immune and metabolic status of the host. microbiome. The studies outlined below strongly suggest that there is
While most mechanistic studies have focused on the effect of spe- indeed a connection and that TLR2 signaling is the key that ties MS to
cific bacterial species and bacterial products on the local (i.e., GI) im- both the hygiene hypothesis and the microbiome. We next present four
mune system, a more difficult and potentially more important question “proof of concept” studies that we believe support this paradigm in MS.
is how the GI microbiome mediates effects on the immune system
outside of the GI tract, i.e., on the entire systemic immune system. At
present, only a small number of studies have suggested mechanisms by 7. First proof of concept: A microbiome-derived PAMP is lower is
which the GI microbiome can affect the systemic immune system. For MS blood
example, it has been reported that specific immune cells, (e.g., Tregs),
initially interacting with and potentially programmed by the micro- In 2013, while studying a family of unique bacterial lipopeptides
biome while in the GI tract, can subsequently leave the GI tract and (“serine lipids”) derived from bacteria in the oral and GI microbiome,
mediate functions in the systemic immune system [31–33]. This me- (produced by several Bacteroides species [40]), we asked whether these
chanism describes an effect of the local environment (GI microbiome) lipopeptides had the ability to be recognized by human immune cells
that then bridges to a more systemic effect via trafficking of cells out of and whether these lipopeptides left their microbiome site and entered
the local environment. A second mechanism involving entry of micro- the human systemic circulation. We found and reported that this family
biome-derived PAMPs into the systemic circulation is discussed in de- of lipopeptides, and specifically one family member, Lipid 654 (L654)
tail below. (Fig. 1A), has the ability to stimulate innate immune cells through TLR2
[40]. Next, to investigate whether L654 can leave its microbiome sites
5. Multiple sclerosis and the microbiome and enter the human systemic circulation, we analyzed human serum
samples for L654. This lipidomic analysis used sophisticated mass
Over the past five years, extensive research has focused on the mi- spectrometry analysis (multiple reaction monitoring [MRM] mass
crobiome in MS. Numerous studies have catalogued the GI bacterial spectrometry). MRM-mass spectrometry allows for unequivocal
populations in patients with MS versus controls. While most studies find
that MS patients have alterations in the composition of their GI mi-
crobiome compared to healthy controls (GI microbial dysbiosis), overall
there has been little consensus in identifying MS-specific microbiome
profiles.
Jangi et al. reported that alterations in microbiome bacterial po-
pulations in MS include increases in Methanobrevibacter and
Akkermansia and decreases in Butyricimonas. Furthermore, these in-
vestigators found that patients on disease-modifying treatment show
increased abundances of Prevotella and Sutterella, and decreased
Sarcina, compared with untreated patients. They concluded that “fur-
ther study is required to assess whether the observed alterations in the
gut microbiome play a role in, or are a consequence of, MS pathogen-
esis” [34]. Chen et al. found an increased abundance of Pseudomonas,
Mycoplana, Haemophilus, Blautia, and Dorea genera in MS patients,
whereas the control group showed increased abundance of Para-
bacteroides, Adlercreutzia, and Prevotella genera. These authors con-
cluded that MS patients have GI microbial dysbiosis and that further
study was needed to understand the role of this dysbiosis in the pa-
thogenesis of MS [35]. Calvo-Barreiro et al., reviewed potential func-
tional links between microbiome alterations and MS, specifically ex-
amining immunoregulatory effects of microbiome changes reported for
RRMS patients [36]. These authors point out that bacterial species Fig. 1. Lipid 654 (L654) is a bacterial-derived lipopeptide found at lower serum
belonging to the Clostridium genus, found in some studies to be un- concentrations in MS patients. (A). Structure of Lipid 654. (B). L654 in Serum.
derrepresented in RRMS patients, are known to be producers of short Serum lipids were derived from 17 MS patients and 13 healthy controls and
chain fatty acids. Short chain fatty acids are reported to promote reg- analyzed for L654 by MRM-mass spectrometry. Results are expressed as ion
ulatory T cells and enhance the production of IL-10 in studies of EAE abundance, Log base 2. [41].

3
N.J. Wasko, et al.
identification and quantification of L654, and through its unique bac-
terial-associated structures, allows it to be differentiated from lipids of
mammalian (human) origin.
Analyzing serum samples from healthy individuals, we were sur-
prised to identify L654 in all serum samples studied. This suggested that
this microbiome-derived TLR2-stimulating bacterial PAMP was routi-
nely and chronically circulating in everyone's bloodstream along with
the “normal” mammalian lipids. Most importantly, when we compared
the serum samples from healthy individuals with those of MS patients,
we found that healthy individuals demonstrated levels of L654 that
ranged from high to low values while, in contrast, MS patients had
levels of L654 that were clustered only in the low range [41] (Fig. 1B).
This finding was striking and initially surprising given that MS is an
autoimmune disease associated with inflammation of the CNS. Because
L654 has the capacity to stimulate cells in a pro-inflammatory manner
via TLR2, we expected serum levels of L654 to be equal or higher in MS
patients compared to controls. However, we were also aware that TLR2
signaling results not only in an initial release of pro-inflammatory cy-
tokines but also induces a state of TLR2 tolerance in which subsequent
TLR2 responses are dampened or inhibited.
8. TLR2 signaling in MS and Experimental Autoimmune
Encephalomyelitis (EAE)
Our findings with L654 prompted us first to ask what role, if any,
does TLR2 signaling play in the pathophysiology of MS. TLRs are a
family of pattern recognition receptors (PRRs) activated by different
PAMPs, which are evolutionarily conserved compounds generated by
bacteria, viruses, fungi, and other infectious species [42,43]. TLRs also
recognize damage-associated molecular patterns (DAMPs) that arise
endogenously from sterile injury or cell turnover [43]. Different TLRs
sense different PAMPs and DAMPs, such as TLR4 that responds to LPS
from Gram-negative bacteria [44] and TLR5 that responds to bacterial
flagellin [45]. TLR2 is capable of being stimulated by triacylated bac-
terial lipopeptides when heterodimerized with TLR1 or diacylated
bacterial lipopeptides when heterodimerized with TLR6 [46], as well as
a wide variety of other non-lipopeptidic PAMPs and DAMPs [47].
When ligated, most TLRs activate the myeloid differentiation pri-
mary-response protein 88 (MyD88)-dependent pathway, with the ex-
ceptions of TLR3, which signals via the Toll/interleukin 1 receptor
(TIR)-domain-containing adapter-inducing interferon-β (TRIF), and
TLR4, which utilizes both MyD88 and TRIF signaling pathways
[42,46,48]. MyD88 activation triggers recruitment of tumor-necrosis-
factor-receptor-associated factor 6 (TRAF6) and members of the IL-1R-
associated kinases (IRAK) family, which allows NF-κB to translocate
into the nucleus [42,46–48]. Nuclear NF-κB increases production of
pro-inflammatory cytokines like tumor necrosis factor α (TNFα) and
other immune mediators like cyclooxygenase-2 [42,47,48].
Studies have reported that the expression of TLR2 is enhanced in
both MS and its murine model, EAE [49–51]. While the role of TLR2
signaling in the pathophysiology of MS is somewhat unclear, overall the
literature supports a disease-promoting role for TLR2 signaling
[52–56]. Key studies include the report that TLR2-deficient mice de-
velop attenuated EAE [50,52,54–56] and that TLR2 signaling inhibits
CNS remyelination [51], a defect seen in patients with MS. Further-
more, there is some evidence that endogenous TLR2 ligands, primarily
in the form of DAMPs, account for the disease-promoting role of TLR2
signaling in EAE and MS [57–59]. Support for this concept includes
documentation that inhibiting DAMP signaling through the use of an-
tibodies to DAMPs can attenuate EAE [58,59]. Expression of DAMPs is
sporadically enhanced when tissue damage or inflammation is present,
thus providing a source of endogenous TLR2 ligands to mediate this
disease-promoting effect.
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Autoimmunity Reviews 19 (2020) 102430
9. TLR tolerance
Evolutionarily, the fact that the expression of TLR2 is highly con-
served signifies the importance of TLR2 signaling in immunity to in-
fections. However, as discussed above, evidence from a number of la-
boratories has documented that TLR2 signaling plays a critical disease-
related role in the pathophysiology of MS. These reports, together with
our documentation that a large percentage of MS patients have sig-
nificantly enhanced responses to TLR2 ligands [60] (see below), sug-
gests a novel therapeutic goal. In this goal, TLR2-responsiveness in
patients with MS would be “tuned-down” to levels equal to or less than
that seen in healthy individuals, while still maintaining the response
level required for protection against infectious agents. This therapeutic
goal would be difficult to achieve with most inhibitor-based (e.g., an-
tibody) or genetic interference-based approaches that tend to be “all or
none” in their inhibition. However, through evolution, biology has
developed its own “tuning-down” mechanism termed “TLR tolerance”
[61–63]. In TLR2 tolerance, cells down-regulate TLR2 responsiveness
after being exposed repeatedly to an adequate TLR2 stimulus. As dis-
cussed below, in recent proof of concept studies, we document that TLR
tolerance can be harnessed to treat autoimmune disease.
TLR tolerance (originally termed “endotoxin tolerance”) is a phe-
nomenon in which prior exposure to TLR-stimulating PAMPs results in
a transient state of refractoriness to subsequent challenge [61]. Phe-
notypically, TLR tolerance is associated with increased phagocytic
ability coupled with reduced antigen presentation in macrophages [64],
as well as decreased expression of pro-inflammatory cytokines like
TNFα and increased expression of anti-inflammatory cytokines like
interleukin-10 (IL-10) [65]. Intracellularly, TLR tolerance induces up-
regulation of negative regulators of MyD88 signaling, including IRAK-
M, A20, BCL3, and microRNAs that target parts of the MyD88 pathway
[65–67]. Because many TLRs converge on the MyD88 signaling
pathway, induction of tolerance to one TLR ligand can induce cross-
tolerance to ligands that stimulate other TLRs [67,68]. TLR tolerance is
well documented in monocytes where repeated ligation of TLRs results
in subsequent signal dampening and a state of TLR tolerance that pre-
vents successive ligations from signaling normally and changes down-
stream effector molecules to a more regulatory phenotype [61,65]. TLR
tolerance has also been demonstrated in other cell types including en-
dothelial cells [69] and, as noted above in studies of asthma and A20, in
lung epithelial cells [4].
10. Our central hypothesis
With the concept of TLR2 tolerance in mind, in 2013 [41] we pos-
tulated that the presence of L654 in all serum samples we studied,
coupled with the findings of lower levels of L654 in MS serum suggested
that: 1) relatively small amounts of L654 (and potentially other mi-
crobiome-derived PAMPs) chronically enter the systemic circulation
from the microbiome; 2) circulating L654 interacts on a chronic basis
with TLR2 on innate immune cells and, at levels found in healthy in-
dividuals, induces a relative state of TLR2 tolerance in the innate im-
mune system; 3) this normal state of TLR2 “tolerance” is not sufficient
to completely inhibit TLR2 signaling, but rather serves as a micro-
biome-driven regulator, dampening or “rheostating down” the magni-
tude of systemic TLR2 responses without completely inhibiting TLR2
responses; 4) this microbiome-driven regulation of TLR2 responsiveness
has evolved to protect the host from over-reacting to endogenous TLR2
stimulants (e.g., DAMPs), while preserving the ability to respond in an
appropriate pro-inflammatory manner to the strong-signaling and high
concentration of TLR2 PAMPs present during an infection; 5) the low
level of L654 found in the serum of MS patients suggests that one
normal function of the microbiome - i.e., to seed the systemic circula-
tion with levels of L654 that are adequate to “rheostat” down the level
of response to TLR2 - is abnormal in MS; and 6) the subsequent en-
hanced responsiveness to endogenous TLR2 stimulants, including
N.J. Wasko, et al.
DAMPs, plays an integral role in the inflammatory and defective re-
myelination pathophysiology of MS (Fig. 2).
11. Recent literature support for our 1st proof of concept and
central hypothesis
Our hypothesis posits that the microbiome is a critical component of
our “environment” and as such, plays an important role as a source of
environmental PAMPs in the context of the hygiene hypothesis. Put
forth in 2013 [41], our central hypothesis gained support in the 2016
report documenting that differences in the function of the GI micro-
biome-derived PAMP, LPS, could alter the incidence of the autoimmune
disease type 1 diabetes (T1D) [6].
In this study, Vatanen et al. studied the GI microbiome of infants
from Finland and Estonia and compared them with the GI microbiome
of infants from nearby Russia [6]. Finland and Estonia are known to
have a significantly higher incidence of allergy, autoimmunity, and
early onset T1D compared to the nearby region of Russia. These in-
vestigators analyzed the microbiome of these subjects and found dif-
ferences in the microbiome bacterial composition between the Finnish/
Estonian subjects and the Russian subjects. Importantly, they found that
the Finnish/Estonian microbiome-derived LPS differed in structure and
function from the Russian microbiome-derived LPS. The LPS produced
by the Russian (low incidence of autoimmunity) microbiome func-
tioned efficiently in inducing TLR4 tolerance. In contrast, the LPS de-
rived from the Finnish/Estonian (higher incidence of autoimmunity)
microbiome was incapable of inducing TLR4 tolerance. Additionally,
when tested in the diabetes-prone NOD strain of mice, the Russian LPS
induced TLR4 tolerance in vivo and inhibited the onset of diabetes,
while the Finnish/Estonian LPS exhibited neither of these effects. Based
on their in vitro TLR-stimulation results, their NOD mice studies, and
the published hygiene hypothesis-related asthma studies, Vatanen et al.
postulated that PAMPs emanating from the microbiome “environment”
have the capacity to “tolerize” cells of the innate immune system and
thereby down-regulate enhanced immune responses underlying auto-
immune and allergic diseases. This study concluded that lack of ex-
posure to microbiome-derived LPS capable of inducing TLR4 tolerance
resulted in inadequate regulation of systemic innate immune TLR re-
sponses and enhanced autoimmunity in the form of T1D. In this way,
this T1D study paralleled the asthma studies discussed above, finding
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Autoimmunity Reviews 19 (2020) 102430
Fig. 2. Central Hypothesis: in Healthy Individuals: GI micro-
biome-derived PAMPs, such as L654, chronically enter the
systemic circulation at low levels, interact with PRRs such as
TLR2 on immune cells, and mediate a level of TLR tolerance
that serves to “rheostat” down the response to subsequent li-
gation. Through evolution, this level of tolerance has been
selected to diminish the stimulation potentially occurring
through intermittent DAMP expression (and thus decrease the
risk for auto-inflammation and autoimmunity), while preser-
ving an appropriate pro-inflammatory response to the en-
hanced concentration and strength of ligands presented by
infectious agents. In Patients with MS: there is inadequate
seeding of relevant and functional GI microbiome-derived
PAMPs into the systemic circulation resulting in inadequate
induction of innate immune TLR tolerance. This inadequate
level of TLR tolerance allows for a significant pro-in-
flammatory response through intermittent DAMP expression
and stimulation, which in turn increases the risk for auto-
immune diseases such as MS. In addition, the enhanced TLR2
responses result in defective remyelination, as discussed
below.
that a paucity of functionally adequate “environmental” bacterial
PAMPs (dust-derived in the asthma studies; microbiome-derived in the
T1D study) resulted in inadequate regulation of systemic innate im-
mune TLR responses and enhanced autoimmunity.
Overall, this T1D microbiome study supports our central hypothesis,
establishing the microbiome as an “environment” that supplies the
systemic innate immune system with a chronic low-dose of PAMPs that
regulate the immune response and aid in controlling the development
of autoimmune disease.
12. Second proof of concept: Systemic TLR2 tolerance induction
inhibits a T-cell transfer model of EAE
To begin to probe the association of TLR2 tolerance and MS, we
conducted proof of concept studies using EAE [70]. We specifically used
the SJL-proteolipid protein (PLP) model of adoptive transfer (AT) EAE
in which disease is mediated in naïve mice by adoptive transfer of ac-
tivated T cells from PLP-immunized mice. This model avoids using
exogenous adjuvants that contain TLR2 ligands. We found we could
enhance the level of TLR2 tolerance beyond the level theoretically
mediated by the microbiome by administering a low dose of the TLR2
ligand Pam2Cys (P2C) intravenously (i.v.) for as few as three days,
which we termed “TLR2 tolerance induction” [70]. Tolerance was
documented by a diminished serum TNFα response to administration of
a larger dose of a second TLR2 ligand after three days of treatment with
P2C. Most importantly, administering low dose P2C for 14 days sig-
nificantly attenuated AT EAE compared to mice receiving vehicle
control (VC) for 14 days (Fig. 3A). The TLR2-tolerized mice with atte-
nuated EAE demonstrated significantly fewer T cells in the CNS com-
pared to control mice [70]. EAE attenuation was also seen when tol-
erance was induced with L654 instead of P2C. We further documented
that in vivo induction of TLR2 tolerance cross-tolerized to other TLRs,
enhancing the efficiency of this mode of immune regulation [70].
In this EAE proof of concept study, we found that TLR2 tolerance
and EAE attenuation was associated with significant decreases in CD80-
expressing macrophages and pro-inflammatory F4/80+CD11c+ mac-
rophages in the CNS (Fig. 3B). We postulated that one mechanism
underlying the decreased T cell numbers in the CNS is diminished T cell
re-stimulation in the CNS by tolerized antigen presenting cells (APCs).
However, the role of specific CNS APC populations in EAE pathogenesis
N.J. Wasko, et al. Autoimmunity Reviews 19 (2020) 102430

Fig. 3. Administration of low-dose P2C attenuates AT EAE. (A). Naïve SJL/J mice were injected intraperitoneally with 29 × 106 PLP139–151-reactive lymph node
cells on day 0. EAE disease severity was recorded daily for each mouse and compiled as a daily mean disease score for each cohort. Injections of vehicle control (VC)
or 0.35 μg Pam2Cys (P2C) began at day −5 and continued through day +8 post cell transfer; 2 independent experiments are compiled. n = 6–10 per group; disease
incidence was VC: 9/10 and P2C: 1/6; p < 0.0001 Mann-Whitney U. (B). Percentage of spinal cord F4/80+ cells that are positive for CD80 expression; (left: n = 9;
*p = 0.0248 by Student‘s t-test) and percentage of spinal cord mononuclear cells that are F4/80 + CD11c+; (right: n = 13; ****p = 0.0001 by Student ‘s t-test [70].

remains controversial and uncertainty remains regarding which cells
are most critical in presenting antigen to CNS-migrating T cells. In our
study [70], we identified alterations in CNS macrophage populations
but not in microglial or classic dendritic cell populations in TLR2-tol-
erized mice. We concluded that the decrease in CNS T cells and CNS
APC activation markers supports the working hypotheses that TLR2
tolerance induction inhibits a stage of disease relating to T cell-APC
interactions affecting T cell re-stimulation and thus T cell survival and
proliferation in the CNS.
stimulation [60]. Interestingly, the enhanced TLR2 responders included
a significant fraction of those with progressive forms of MS, a subset of
patients considered largely unresponsive to adaptive immune system-
targeting therapies (Fig. 4A and B). The results of this study are con-
sistent with our central hypothesis suggesting that there is defective
regulation of TLR2 responses in patients with MS and further highlight
the presence of a pathologically relevant TLR2-related innate immune
abnormality in patients with both relapsing-remitting and progressive
forms of MS.

13. Third proof of concept: enhanced TLR2 responses in multiple
sclerosis
Based on the lower levels of serum L654 in MS patients (first proof
of concept), the therapeutic efficacy of TLR2 tolerance in EAE (second
proof of concept), and our hypothesis that TLR2 tolerance is defective
in MS patients, we next postulated that defective TLR2 tolerance will
result in TLR2 hyper-responsiveness in MS patients. Studies of TLR
responses in patients with MS have been conflicting. Importantly, most
of these investigations have focused on the response to TLR4 ligation
and few have characterized TLR2 responses in MS. In this third proof of
concept study, our goal was to use multiple approaches to characterize
TLR2 responses of peripheral blood mononuclear cells (PBMCs) and
CD14+ monocytes derived from MS patients. Studying a total of 26 MS
patients and 32 healthy controls, we documented for the first time that
50% of MS patients demonstrate enhanced responsiveness to TLR2
14. Fourth proof of concept: systemic TLR2 tolerance induction
enhances remyelination in the cuprizone model of CNS
demyelination
MS involves not only an autoimmune CNS inflammatory process but
also a defect in the ability to remyelinate the neurons demyelinated in
this inflammatory process [14,71,72]. It is known that in MS there is a
loss of the myelin-producing cells, the oligodendrocytes (OLs) [73].
Although demyelination can theoretically be repaired by differentiation
of oligodendrocyte precursor cells (OPCs) into mature OLs, for reasons
as yet unknown, remyelination is defective in MS [71,73–75], and di-
minished differentiation of OPCs into mature OLs may contribute to this
defect in remyelination [14,71,72].
Enhancing remyelination has become an increasingly important
goal of MS research. While studies have suggested that signaling
through TLR2 plays a critical role in the inflammatory pathogenesis of

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N.J. Wasko, et al. Autoimmunity Reviews 19 (2020) 102430

Fig. 4. MS patients exhibit enhanced TLR2 responsiveness compared to healthy controls. (A). CD14+ monocytes from MS patients (15 patients; 16 total responses)
and healthy controls (13 individuals; 15 total responses), were cultured at 105/ml, stimulated with 2 μg/ml of Pam 2Cys (P2C) or Pam3Cys (P3C) for 4 h, and TNFα in
the culture supernatants was measured by ELISA. The level of TNFα representing the upper threshold of control responses [based on the control interquartile range
(IQR)] is depicted by the dashed line. The percentage of responses above the threshold is depicted for control and total MS patients. Medians are depicted. (a) Red
icons represent P2C stimulation and black icons represent P3C stimulation; (b) same results as in (a), but green icons represent patients with progressive MS. The
mean TNFα level for the control cohort was 104 ρg/ml and the mean TNFα level for the total MS cohort was 247 ρg/ml; P = 0.0712 via Mann–Whitney. The
percentage of responses above threshold: 57% of progressive MS patients, 33% of RRMS patients. (B). Compilation of percentages of control and MS patients with
enhanced TLR2 responses as assessed in 3 studies using: peripheral blood mononuclear cells (PBMC), CD14+ whole population, or CD14+ single-cell analyses. These
compiled studies encompass 26 MS patients and 32 healthy controls. The percentages of progressive MS patients (Prog MS) with enhanced responses
(mean = 60.13%) were significantly greater than those of the control patients (mean = 6.87%); P = 0.0098 via Kruskal–Wallis followed by Dunn's multiple
comparisons test. The percentages of total MS patients (51.55%) and RRMS patients (41.65%) with enhanced responses were greater than those of the control
patients, but did not reach statistical significance. Medians are depicted [60].

MS and EAE [70,76,77], only few studies have investigated a role for
TLR2 signaling in remyelination. Critically, the majority of these studies
have demonstrated an inhibitory effect of TLR2 signaling on re-
myelination [51,78,79]. Prior studies have reported that the in vitro
maturation of OPCs to mature OLs is inhibited by TLR2 ligation, and
that hyaluronate, an endogenous TLR2 ligand (i.e., a DAMP), may be a
relevant mediator of this inhibition of remyelination [51]. Furthermore,
mice in which TLR2 was genetically deleted (TLR2−/− mice) demon-
strated enhanced remyelination in vivo after lysolecithin-induced de-
myelination [51].
Based on studies relating TLR2 signaling to the inhibition of re-
myelination [51,78,79] and the potential for therapeutic intervention
in EAE and MS via TLR2 tolerance induction [60,70], we next tested the
role of TLR2 tolerance induction in the process of CNS remyelination.
Since we previously documented that TLR2 tolerance induction could
attenuate the CNS inflammation in EAE [70], in this study we used the
non-inflammatory cuprizone model of demyelination [80].
Mice were fed cuprizone for five weeks to induce a non-in-
flammatory demyelination, followed by a two-week feeding with
normal chow to allow for remyelination. We asked whether diminishing
TLR2 signaling by inducing systemic TLR2 tolerance during the two-
week recovery period would enhance remyelination. TLR2 tolerance
was induced by daily administration of low dose P2C beginning when
the cuprizone feeding was stopped and continuing for the two-week
recovery period. Results showed that inducing TLR2 tolerance during
the recovery period resulted in a significant enhancement in re-
myelination as documented by electron microscopy (EM) evaluation of
both percentage of unmyelinated axons and myelin thickness (g-ratios:
the ratio of the inner axonal diameter to the total outer diameter) [81]
(Fig. 5A, B). As shown in Fig. 5 A–B, results based on evaluation of the
EM images demonstrated that the percentage of unmyelinated axons
was significantly less in the TLR2 tolerized mice and the myelin
thickness was significantly greater in the TLR2 tolerized mice (evi-
denced by a lower g-ratio). In fact, at the two week time period of
recovery, the degree of remyelination in TLR2 tolerized mice was
equivalent to that normally seen only after four-to-five weeks of re-
covery in control mice, and was identical to that seen in mice that had
never undergone demyelination.
In confirmation of these results, we documented that TLR2−/− mice
showed a significant enhancement in remyelination using the same EM
evaluation parameters [81]. In line with previous studies [78], TLR2−/
−
mice lost myelin at levels similar to WT mice when examined im-
mediately after five weeks of cuprizone exposure. However, when given
two weeks to recover on normal chow (without any intervention),
TLR2−/− mice exhibited a significant enhancement in remyelination,
phenocopying the effect seen in TLR2-tolerized WT mice [81]. These
results suggest that TLR2 tolerance induces a state of functional TLR2
deficiency, allowing myelin to recover without the interference medi-
ated by endogenous TLR2 signaling.
Using immunohistofluorescent approaches, we found that the TLR2

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N.J. Wasko, et al. Autoimmunity Reviews 19 (2020) 102430

Fig. 5. Induction of TLR2 tolerance enhances remyelination during recovery from cuprizone feeding. (A) Percentage of unmyelinated axons and (B) myelin thickness
(g-ratios) as calculated from EM images. VC: mice treated with vehicle control during recovery; P2C: mice treated with the TLR2 ligand, P2C. during trecovery. Data
points represent individual mice. N = 8–10 mice per experimental condition. Statistical differences were assessed by 2-way ANOVA. (C) Percentage of IBA1+ cells
that are iNOS+, Arg1- (D) Percentage of IBA1+ cells that are Arg1+, iNOS-. Error bars represent the mean ± SEM. Statistical differences were assessed by 2-way
ANOVA, * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (adapted from [81]).

tolerance-induced enhancement in remyelination was not associated
with changes in the number of OPCs or mature OLs, but rather was
associated with a change in the balance of microglial (IBA1+) pheno-
types from pro-inflammatory M1 (iNOS+ Arg1−) to non-inflammatory
M2 (iNOS− Arg1+) [81] (Fig. 5C, D). Functionally, microglia (like
macrophages) can be divided into different states of activation. Though
likely an oversimplification, M1 microglia are described as “pro-in-
flammatory” with regards to their cytokine secretion while M2 micro-
glia, or “alternatively activated” microglia, are associated with extra-
cellular matrix remodeling, progenitor cell differentiation, and tissue
regeneration [82–84].
These results suggest that the enhanced remyelination seen with
TLR2 tolerance results, at least in part, from effects on microglial ac-
tivation phenotype, and specifically on an increase in the percentage of
microglia expressing an M2 phenotype. While there is not yet a clear
understanding of how M2 microglial polarization results in enhanced
remyelination, proposed mechanisms include facilitating OPC to OL
differentiation [85,86] and increasing phagocytic clearance of myelin
debris [87,88]. In sum, this study presents evidence that the innate
immune system, and TLR2 specifically, has a role in the process of re-
myelination. Moreover, together with our previous findings of lower
serum L654 levels in MS patients [41], TLR2 tolerance-induced at-
tenuation of EAE [70], and enhanced in vitro TLR2 responsiveness in
50% of MS patients [60], the results of this remyelination study further
support our central hypothesis that inducing TLR2 tolerance in vivo
mediates functional changes in TLR2 signaling that have physiologic
and disease-relevant effects. By demonstrating the relevance of TLR2
signaling in remyelination, the present study also confirms earlier stu-
dies suggesting that TLR2 signaling plays a critical role in the re-
myelination defect noted in patients with MS.
15. Summary and conclusions
Many components of the pathophysiology of MS remain unclear.
Although extensive research has focused on the relationship between
autoimmune diseases and the hygiene hypothesis or autoimmune dis-
eases and the microbiome, as yet no conclusive breakthroughs in our
understanding of disease mechanisms in MS have come from these
approaches. In this review, we present a novel paradigm that connects
the pathophysiology of MS to both the hygiene hypothesis and the
microbiome.
A key to our paradigm is the concept that abnormally enhanced
TLR2 signaling is a critical player in both the autoreactive CNS in-
flammation and defective remyelination seen in MS. An abnormally
enhanced TLR2 responsiveness is, in turn, dependent on both a mi-
crobiome that is defective in providing adequate TLR2-tolerizing

8
N.J. Wasko, et al.
PAMPs to the systemic immune system and the hygiene hypothesis that
posits that environmental PAMPs are required to adequately regulate
and “tune” innate immune responses. In terms of the hygiene hypoth-
esis and MS, the microbiome is the relevant “environment” and its
defective delivery of “environmental” PAMPs to the systemic immune
system makes it functionally “too clean.” The result is innate immune
over-responsiveness that we postulate is critical to the pathophysiology
of MS.
In outlining our postulate, we present four proof of concept studies
that support the inflammatory component (EAE studies), the re-
myelinating component (cuprizone studies), and the human immune
response component (serum L654 levels and peripheral blood monocyte
TLR2 responses) of our paradigm. It should be noted that we do not
believe the one PAMP we have studied in MS, L654, is necessarily the
only or most relevant circulating PAMP in this paradigm. However,
Calvo-Barreiro et al. note that L654 is produced by Bacteroides species
and the proportion of several Bacteroides genera is reported to be re-
duced in the microbiome of RRMS patients, suggesting that “…defi-
ciency of this naturally occurring innate immune regulation could lead
to the escape of autoreactive immune responses and could represent a
mechanism linking the commensal microbiome and MS…” [36]. We
believe that our proof of concept studies lay the foundation for con-
tinued documentation of the relevance of this paradigm in MS and also
for more basic studies confirming the relationship between the micro-
biome production of systemically “tolerizing” PAMPs and innate im-
mune regulation in health and disease.
Finally, our studies to date highlight the dual role of TLR2-signaling
in autoimmune CNS inflammation and defective CNS remyelination,
and suggest that induction of TLR2 tolerance may represent a novel
two-pronged approach to treating MS, inhibiting autoimmune in-
flammation while simultaneously facilitating myelin repair.
Funding source
Connecticut Bio Innovations Fund (CBIF, Grant #517) funded this
research. CBIF had no role in study design; in the collection, analysis
and interpretation of data; in the writing of the report; and in the de-
cision to submit the article for publication.
Declaration of Competing Interest
None.
This manuscript is not under consideration for publication else-
where, that its publication is approved by all authors and tacitly or
explicitly by the responsible authorities at the University of Connecticut
Health Center, and if accepted, it will not be published elsewhere in the
same form, in English or in any other language, including electronically
without the written consent of the copyrightholder.
Acknowledgements
The authors would like to thank Dr. Glenn Matsushima, University
of North Carolina School of Medicine, for his expert advice on using the
cuprizone model, Ms. Maya Yankova of the UConn Health Electron
Microscopy Core, and Dr. Evan Jellison of the UConn Health Flow
Cytometry Core for their expert technical assistance. We also thank all
the healthy volunteers and the MS patients for their participation in our
study as well as the staff of the Lowell P. Weicker Jr. Clinical Research
Center, UConn Health.
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