Unraveling the Hygiene Hypothesis of helminthes and autoimmunity: origins, pathophysiology, and clinical applications

For several decades, Western countries have been facing an increasing incidence of
allergic and autoimmune disorders 1]-6]. Thus, in the United States (US), the prevalence of asthma in children has increased
by 38% between 1980 and 2003. Similarly, this rise has reached 56% and 59% between
1964 and 1990 in Scottish and Australian children, respectively 1]. Regarding autoimmune diseases, the incidence of inflammatory bowel disease (IBD)
has risen in the past 50 years up to 8 to 14/100,000 persons for ulcerative colitis
(UC) and 6 to 15/100,000 persons for Crohn’s disease (CD) 4]. Likewise, the annual increase in incidence of type-1 diabetes (T1D) ranged from
2.9% to 5.4% per year according to the countries during the 1989–2003 period 3]. Also, the number of people living with multiple sclerosis (MS) worldwide has climbed
from 2.1 million in 2008 to 2.3 million in 2013 6]. Quoted here are only few examples illustrating the major public health problem posed
nowadays by this epidemic.

Immune-mediated conditions are thought to result from a complex interplay between
genetic predisposition, immune dysregulation, and environmental factors 7],8]. Since genetic basis has not undergone any major changes in such a short period of
time, environmental factors are highly suspected to be responsible for this recent
outbreak. Especially, vitamin D deficiency 9], tobacco 10], air pollution 11], adjuvants 12], and obesity 13] have been incriminated in the pathogenesis and the recent rise in chronic inflammatory
disorders. Together with these factors, infections are widely demonstrated to play
a critical role in autoimmunity 14]. The so called Hygiene Hypothesis (HH) postulates that the reduced exposure to microorganisms
in industrialized countries resulting from improved sanitary conditions would increase
immune reactivity, thus promoting the development of allergic and autoimmune diseases
15]. From its first formulation in 1989 16], this theory has been strengthened by solid epidemiological, experimental, and clinical
data, paving the way for future therapies.

In the first part of this opinion piece we discuss epidemiological data, originally
from allergic diseases followed by autoimmune diseases, which have given birth and
reinforced the concept of the HH. Since these abundant epidemiological observations,
numerous experimental studies have clarified the type of microorganisms involved in
this theory, thereby specifying their immunoregulatory effects. Thus, a wide spectrum
of viruses, bacteria – especially from the gut microbiome – and parasites have been
implicated as major actors in this theory. Therefore, there is now a growing and exciting
literature regarding the key role played by these microbes in both allergic and autoimmune
disorders 17]-20].

Herein, we have chosen to focus on the emerging role of parasites, particularly helminthes
in autoimmune diseases. First, we will specify their immunoregulatory effects in autoimmune
diseases identified through experimental models. Then, we will detail the experimental
studies from animal models and the first clinical trials conducted in humans that
have resulted from the HH.

Birth of the Hygiene Hypothesis (HH)

The ability of infectious agents to modulate the immune system has long been a fascinating
topic. For almost half a century, infections have been widely demonstrated to act
as triggering factors of autoimmune response. Viral agents, such as Epstein-Barr virus,
cytomegalovirus, and parvovirus B19, as well as numerous bacteria and parasites have
been associated with the presence of a wide variety of autoantibodies and found to
contribute to the pathogenesis of autoimmune diseases 14],21]. Thus, parasitic infections have been shown to promote autoimmunity through various
mechanisms, including molecular mimicry of parasitic epitopes, alteration of host
antigens, polyclonal activation, and expansion of autoreactive B-cell clones, as well
as manipulation of the idiotypic network 22].

At the same time, it became increasingly evident that infectious microorganisms could
also exert an immunomodulatory and immunodepressant action on the immune system, resulting
in a protective effect against immune-mediated conditions such as allergies and autoimmune
diseases. The changes that have accompanied the recent modernization in Western countries,
such as migration from rural to urban areas, improved sanitation, access to clean
water, control of food production, or even vaccination campaigns, have reduced contact
with these ancestral microorganisms with which mammals had coexisted and co-evolved
for millennia 23]. Hence, the HH concept emerged, postulating that the slightest exposure to these
immunoregulatory infectious agents – called ‘Old Friends’ by Rook 24] – in industrialized countries due to improvement in hygiene conditions promotes the
development of chronic inflammatory disorders and contributes to their recent rise
15].

This theory was first suggested by Greenwood 25], nearly half a century ago, when reporting a lower prevalence of autoimmune diseases
in Nigerians. He suggested an immunomodulatory effect of multiple parasitic infections
since childhood, later confirmed by demonstrating that infection of different strains
of mice prone to developing autoimmune diseases with the rodent malaria parasite Plasmodium berghei prevented the occurrence of the disease 26]. The inverse correlation between the dramatic decrease in infections in industrialized
countries due to better hygiene and the concomitant increase in immune-mediated diseases
was finally clarified by Strachan in 1989 16]. Indeed, by following a cohort of more than 17,000 children born in 1958 for 23 years,
he observed an inverse relationship between the number of older siblings in the household
and the prevalence of hay fever, therefore concluding that allergies could be prevented
by infections in early childhood. According to Strachan, a lower exposure to these
infections might promote atopic diseases. These observations have led to the birth
of a new paradigm on the role of infectious agents in immune disorders. Since then,
the HH has been widely powered by epidemiological, experimental, and clinical data.

Epidemiological evidence

The HH, as formulated by Strachan a quarter-century ago 16], originally focused on allergic diseases. It claimed that their recent rise in Western
countries was promoted by reduced exposure to microorganisms due to improved hygiene
conditions. Since these early observations, many epidemiological data have reinforced
this theory, first on allergic disorders and then extending to autoimmune diseases.

A number of studies have investigated the prevalence of allergic diseases according
to living conditions. First, the initial observation of Strachan 16], demonstrating an inverse correlation between the sibship size and the subsequent
risk of allergy, has since been widely replicated in a large number of studies in
affluent countries 27]-30]. Moreover, Strachan et al. 31] recently confirmed, in a broad international study involving more than 500,000 children
in 52 countries, the inverse association between the risk of developing hay fever
or eczema and the total number of siblings; the association being stronger in more
affluent countries. Otherwise, pet ownership has also been linked to a decreased prevalence
of allergic diseases. In a recent meta-analysis including 36 publications, Pelucchi
et al. 32] reported a favorable effect of exposure to pets, especially to dogs, on the risk
of atopic dermatitis in infants or children. Similarly, worst living standards in
Eastern Germany compared with Western Germany were associated with a reduced occurrence
of atopic diseases 33]. Thereafter, the prevalence of atopy experienced an increase in children in Eastern
Germany born after the reunification of Germany in 1990 34]. Equally, other lifestyle factors, including low antibiotic consumption 35],36] and growing up in rural areas, were associated with a diminished prevalence of allergic
diseases 37],38]. In developing countries, an inverse relationship was also observed between the prevalence
of parasitic infections, especially helminthic, and the risk of allergic diseases.
For example, in Ecuador 39], Gabon 40], and Brazil 41], helminth infections were shown to have a protective effect on allergic reactivity.
Conversely, anti-helminthic treatment of chronically infected children in Gabon 40], Venezuela 42], and Vietnam 43] resulted in increased atopic reactivity.

The HH was later extended when the protective effect of infectious agents, especially
parasites, against autoimmune diseases was suggested through various epidemiological
studies 15]. As previously reported, the number of siblings has been shown to correlate inversely
to the risk of MS 44],45]. Furthermore, in the Italian island of Sardinia, several epidemiological and immunogenetic
evidences 46]-49] link malaria eradication 50 years ago with the concomitant increase of MS. It is
assumed that the strong genetic selective pressure of malaria along the centuries
led to the selection of polymorphisms and genotypes conferring resistance to Plasmodium falciparum, the causative agent of malaria. These polymorphisms are responsible for the increased
immune reactivity required to control malaria. As soon as the immunoregulatory organism
was withdrawn from the environment by the modern lifestyle, these genetic variants
led to excessive inflammation and became susceptibility factors for chronic inflammatory
disorders, especially MS.

The Karelia region is also of great interest to investigate the influence of lifestyle
and infections on the risk of immune-mediated disorders. This region is divided into
a Finnish area, characterized by high standards of hygiene, and a Russian area, with
poorer hygiene and an increased rate of infections. Finnish Karelian maintain one
of the highest prevalence of autoimmune and allergic diseases, while Russian Karelian
prevalence is far lower, despite sharing the same genetic background. For example,
the incidence of T1D is six-fold higher in Finland compared to the adjacent Karelian
republic of Russia, the wide gap in infection rates between the two regions being
strongly suspected to contribute to this difference 50],51]. Subsequently, Weinstock et al. 52] expanded the HH to IBD based on the increasing prevalence of IBD in the US contrasting
with the declining prevalence of helminthes. This observation has since been confirmed
in other parts of the world. In sub-Saharan Africa, where helminthic infestation is
frequent, a low incidence and prevalence of IBD is observed which cannot be explained
by genetic factors due to the fact that, in black populations of the US and UK, the
incidence of IBD is approaching that of the white populations 53]. Moreover, migration studies have shown that descendants of immigrants coming from
a country with a low incidence acquire the same incidence as the host country, as
illustrated for T1D 54],55] and MS 56],57]. Similarly, the prevalence of systemic lupus erythematosus (SLE) was found to be
much higher in African Americans compared to West Africans 58]. Interestingly, a case–control study in India showed that none of the patients with
rheumatoid arthritis (RA) were positive for circulating filarial antigen in contrast
to 40% of healthy controls 59].

Such epidemiological observations raise questions concerning the nature of the protective
infectious agents involved and the mechanisms through which they modulate the immune
system and thus the risk of inflammatory disorders.

These epidemiological data, in conjunction with other data, reinforced the paradigm
that infectious agents may confer a protective effect against chronic inflammatory
diseases. Therefore, it is of interest to unravel and clarify the mechanisms of this
theory.

Pathophysiology of the Hygiene Hypothesis (HH)

Who are the actors in the HH?

The term ‘Hygiene’ in the HH refers to all changes in our lifestyle corresponding
to ‘Westernization’ and resulting in greater hygiene levels. These developments have
affected, in particular, our living space, food preparation, access to clean water,
and medical and therapeutic care, and have thereby led to profound changes in our
microbial environment. Therefore, beyond these general concepts of Hygiene and Westernization,
it is critical to understand and clarify exactly which germs the recent modernization
has removed from our ‘microbiome’ (i.e., microbes we are in contact with). Consistent
with the HH, it would be the disappearance of these organisms from our environment,
depriving us of their immunomodulatory properties, which would have contributed to
the recent outbreak of immune-mediated disorders in Western countries.

The evolution of our microbial environment through the millennia was elegantly analyzed
by Rook 24],60], who refers to these organisms as ‘Old Friends’. According to him, the relevant microorganisms
with a probable immunoregulatory role are those that were part of our natural environment
and with which we co-evolved and lived in close contact since periods as far back
as the Paleolithic, until a few decades ago, when our society was still largely rural,
living on farms and in contact with animals 61]. So far, we accepted and tolerated these organisms in our body, cohabiting in relative
harmony with them, the latter being ultimately largely removed from our modern urbanized
environment. In view of this, we are primarily concerned with helminthes and microbes
acquired by oro-fecal transmission or that can induce an asymptomatic carrier state
(hepatitis A virus, Mycobacteria, Toxoplasma, Helicobacter pylori) as well as those composing the commensal (cutaneous, intestinal, oro-pharyngeal,
genitourinary) and the environmental flora (present in mud, water, soil, plants, animals).
Common infections of childhood were often considered as part of the relevant microbes
in the HH. However, most childhood viruses, such as measles, mumps, and chickenpox,
are most frequently not protective against chronic inflammatory disorders 28],62],63], and even often trigger them 64]. Unlike other previously mentioned microorganisms, these have not peacefully co-evolved
with us, and either have a harmful effect that can kill the host, or induce a strong
immune response. Called by Rook ‘crowd infections’ 60], these viruses require large populations and close contacts to persist, but do not
exert a beneficial role in our organism and therefore, unlike other microbes, did
not coexist with us.

Evolution of our microbial environment in the Western world

It is of interest to attempt to understand what developments in our lifestyle have
led to the dramatic changes in our contact with these ancestral organisms within a
few decades.

First, urbanization and migration of populations to cities, as well as public health
measures, such as control of food production, water quality, and advances in sanitation
and health care, have significantly reduced or almost eradicated some infections in
Western countries, particularly helminthic infections 65],66], malaria, mycobacterial infections 67], and hepatitis A. As indicated above, epidemiological data have inversely correlated
the eradication of some of these infections, especially helminthic infections and
malaria, with an increase in the prevalence of immune-mediated diseases in Western
countries.

Otherwise, the composition of our gut microbiota strongly depends on our environment,
mainly on our microbial contacts as well as many other factors that can modulate it.
It has been demonstrated that the gut microbiota plays a critical role in regulating
the immune response 68],69]. Thus, any factor causing a dysregulation of the microbiota can affect the balance
of our immune system and thereby promote the development of chronic inflammatory diseases
70]-72]. Hence, population migration from rural areas in contact with animals and environmental
flora to more sanitized urban areas has affected the microbiota diversity 73],74], thereby likely favoring immune-mediated disorders. Interestingly, several studies
17],75],76] have suggested that the protective effect of large families or owning animals on
atopic diseases reported in epidemiological studies could be partially related to
an increase in gut microbiota diversity and richness. This may explain the pioneering
observations reported by Strachan 16] on the protective effect of a large number of siblings on the occurrence of allergic
disorders. Similarly, a Western diet 77]-80], widespread use of antibiotics 81], and birth by caesarean section 82] are well-established factors disrupting intestinal microbiota. Several studies have
demonstrated that exposure to antibiotics at an early age may cause dysbiosis, increasing
the risk of subsequent allergic disorders 36],83],84]. In addition, caesarean birth has been associated with a higher risk of asthma 85], T1D 86], MS 87], and celiac disease 88],89].

Thus, eradication of most of these ‘Old Friends’ from our environment may have contributed
to the recent outbreak in inflammatory disorders in Western countries. To better explain
this inverse correlation, it is important to unravel the wide immunomodulatory effects
of these microorganisms on their host’s immune system. To date, helminthes have provided
the greatest information to specify the protective mechanisms developed in the host,
most data being derived from animal models.

An example of immunomodulation by infectious agents: about helminthes

Helminthes are eukaryotic parasitic worms. In 2008, it was estimated that about 37%
of the world’s population was infected with helminthes, mainly in developing countries,
helminthiasis now being rare in industrialized countries 66]. Helminthes’ classification is based on numerous factors, including the external
and internal morphology of egg, larval, and adult stages. These parasites are divided
into two phyla: Platyhelminthes (flatworms), including both trematodes (flukes) and
cestodes (tapeworms), and Nemathelminthes, including only one class, namely nematodes
(roundworms) 90]. Helminthes most frequently live in the gastrointestinal tract of their host, but
may also colonize other organs. It is worth noting that helminthes have co-evolved
with their host for millennia; their goal is not to kill their host but to survive
as long as possible by creating a state of tolerance. To achieve this, helminthes
are able, through various mechanisms, to finely modulate the host immune system to
prevent an activation that may lead to their elimination, while not causing too deep
an immunosuppression which would cause the host to die from infection. This immunomodulation,
by avoiding an excessive activation of the immune system, contributes to host protection
against inflammatory disorders.

Numerous studies in animal models have highlighted the intricate mechanisms by which
helminthes hamper the host’s immune response. This includes promotion of T-helper-2
(Th2) and inhibition of Th1/Th17 differentiation, amplification of T-regulatory (Treg)
and B-regulatory (Breg) cells and type 2-macrophages, orientation of dendritic cells
(DCs) towards a tolerogenic phenotype, downregulation of type-2 innate lymphoid cells
(ILC2), and modulation of the gut microbiota 24],91]-95]. Helminthes’ immunoregulatory effects are illustrated in Figure 1.

Figure 1. Immunoregulatory effects of helminthes on the immune system. Helminthes exert their immunoregulatory actions by modulating cells of both the innate
and adaptive immune system. Regarding T-cells, helminthes may promote a Th2-type response
and down-regulate Th1/Th17 differentiation, leading to increased Th2-type cytokine
(IL-4, IL-5, IL-9, IL-10, IL-13) and decreased Th1/Th17-type cytokine (TNF-?, IFN-?,
IL-6, IL-12, IL-17) secretion. Furthermore, worms’ products enhance Treg cell proliferation,
the latter hampering Th1/Th2/Th17 polarization mainly through the secretion of IL-10
and TGF-?. Helminthes also promote a regulatory phenotype of B-cells, DCs, and macrophages.
Both tolerogenic DCs and regulatory M2-macrophages contribute to switching from a
Th1/Th17 to a Th2/Treg profile. Finally, these parasites may hamper the proliferation
of ILC2, a subset of innate immune cells responsible for allergic responses. Thus,
helminthes create a tolerant environment ensuring their own survival but also protecting
the host from immune-mediated conditions by limiting excessive inflammatory and autoimmune
phenomena. We declare that this figure is original. Breg, B-regulatory cell; DC, Dendritic
cell; IFN, Interferon; IL, Interleukin; ILC2, Type-2 Innate lymphoid cell; TGF, Transforming
growth factor; Th, T-helper cell; TNF, Tumor necrosis factor; Treg, T-regulatory cell.

Th1 and Th2 cells

Naïve T-cells may differentiate into either Th or Treg cells. Regarding Th cells,
the old paradigm opposed two main responses, Th1- and Th2-type responses, down-regulating
each other 96]. Thus, the Th1 response is demonstrated to be particularly involved in the pathogenesis
of autoimmune diseases and is associated with expansion of Th1 cells as well as secretion
of pro-inflammatory cytokines, including interleukin (IL)-6, IL-12, interferon (IFN)-?,
and tumor necrosis factor (TNF)-?. Conversely, the Th2 response plays a central role
in atopic disorders and is characterized by expansion of mast cells, eosinophils,
and increased levels of IL-4, IL-5, IL-9, IL-10, IL-13, and IgE 97]. The original vision derived from experiments in mouse models of autoimmune diseases
infected with helminthes 98] suggested that helminthes were acting on the Th1/Th2 balance by promoting Th2 and
inhibiting Th1 polarization, resulting in a protective effect against Th1-mediated
autoimmune diseases. However, this conception did not explain the epidemiology of
the HH showing a protective effect of helminthes also with regard to allergic diseases.
Indeed, by promoting a Th2 differentiation, helminthes should increase atopic disorders.
Therefore, it has become increasingly obvious that other cellular actors are involved
in immunoregulation mediated by helminthes such as Th17 and Treg cells. Th17 cells
are a recently-defined subset of Th cells primarily secreting IL-17 and whose role
in autoimmune diseases has been widely documented 99],100]. Treg cells 101], for their part, have been found to control both Th2-mediated allergy 102] and Th1/Th17-mediated inflammatory disorders, mainly through the secretion of IL-10
and TGF-? 103].

Many experiments have demonstrated that helminthes are able to modulate the host’s
immune response by inhibiting Th17 differentiation 104]-108], promoting Th2 relative to Th1 polarization 104],106],107],109]-114], and chiefly by enhancing Treg cell proliferation and secretion of IL-10 and TGF-?
104]-107],110]-113],115], resulting in an overall control of Th1/Th2/Th17 responses 92]. The modulation of the T-cell profile contributes to the protective effect of parasites
against both allergic and autoimmune diseases. For example, prior infection of Dark
Agouti rats – prone to developing experimental autoimmune encephalitis (EAE), the
most commonly used animal model of MS – with the nematode Trichinella spiralis (T. spiralis) 104], reduced clinical and histological manifestations of the disease. It was accompanied
by a down-regulation of Th1 (IFN-?) and Th17 cytokines (IL-17) and an up-regulation
of Th2/Treg cytokines (IL-4, IL-10) as well as Treg cell proliferation. Moreover,
transfer of splenic T-cells from T. spiralis-infected rats into non-infected EAE rats led to an improvement of EAE and, in some
cases, protection from disease development. Similarly, the excretory-secretory (ES)
product of the rodent filarial nematode Acanthocheilonema vitae (A. vitae), namely ES-62, was found to suppress collagen-induced arthritis (CIA) severity and
progression in mice by inhibiting Th1- and Th17-associated cytokines (TNF-?, IFN-?,
IL-6, IL-17) 108],114]. Schistosoma mansoni (S. mansoni) infection also significantly reduced the severity of mice CIA by lowering pro-inflammatory
cytokines (IFN-?, TNF-?, IL-17A) and rising Th2/Treg cytokines (IL-4, IL-10) 107].

Dendritic cells

DCs are recognized as a pivotal link between the innate and adaptive immune system.
Their main function is to capture, process, and present antigens to T-cells. Pattern
recognition receptors, including Toll-like receptor (TLR) and C-type lectin receptor
families, are particularly important to this process 116]. The activation status of DCs is crucial in T-cell polarization 117]. Thus, DCs may adopt a ‘tolerogenic’ phenotype opposing the ‘immunogenic’ phenotype,
able to initiate Th2 and Treg responses 118]. These tolerogenic DCs have been shown to be essential in the prevention of autoimmunity
116]. Helminthes’ products are able to modulate DC signaling to direct their differentiation
toward a tolerogenic phenotype 116],119]. This modulation is partly mediated by the binding and recognition of parasitic products
by TLR and C-type lectin receptors 120]. In the Dark Agouti rat model of EAE reported above, the authors reproduced the beneficial
results obtained with T. spiralis infection 104] by injecting non-infected rats with DCs stimulated with ES products released from
encysted muscle larvae of T. spiralis (ES-L1) 7 days before EAE induction 121]. ES-L1-stimulated DCs increased the CD4+ CD25+ Foxp3+ Treg cell population as well
as IL-4, IL-10, and TGF-? production, and decreased IFN-? and IL-17 levels. Further,
two studies 122],123] used a Rag IBD mice model (Rag mice lack functional T- and B-cells) where animals
are reconstituted with IL-10?/? T-cells; IL-10 being a key immunoregulatory cytokine,
IL-10?/??deficient mice develop spontaneous chronic colitis. In these studies, Heligmosomoides polygyrus (H. polygyrus), an intestinal helminth, was demonstrated to prevent and reverse intestinal inflammation,
either by direct infection of IBD mice 122] or by transfer of DCs from infected-mice to IBD mice 123]. Thus, H. polygyrus was shown to mediate IBD protection by altering DC function in a regulatory phenotype.
H. polygyrus-exposed tolerogenic DCs rendered T-cells hyporesponsive and inhibited IFN-?/IL-17
responses.

B-regulatory cells

Helminthes may promote the proliferation of B-regulatory (Breg) cells 124],125]. Besides conventional B-lymphocytes responsible for T-cell activation and antibody
production, a specific subset of B-cells has recently been highlighted. Breg cells
have been shown to negatively regulate the immune response by producing regulatory
cytokines, mainly IL-10, and through direct interaction with pathogenic T-cells. The
regulatory function of Breg cells has since been demonstrated in various pathological
conditions including autoimmune diseases 126],127]. In CIA mice, the ES product of A. vitae, ES-62, in addition to modulating T-cell response as described above, is able to
restore IL-10-producing Breg cell levels while decreasing plasma cell infiltration
in the joints 128]. Similarly, the generation of Breg cells by helminthes was reported in MS 129] and IBD 130] mice models. Interestingly, Correale et al. 131] demonstrated that helminth-infected MS patients created a Breg cell population producing
high amounts of IL-10 as well as neurotrophic factors involved in the growth and development
of neurons. These patients exhibited significantly lower clinical and radiological
disease activity when compared to non-infected patients.

Innate immune cells

Besides modulating T-cells, DCs, and Breg cells, helminthes were also shown to exert
their immunomodulatory effects by manipulating innate immune cells, especially alternatively
activated or M2-macrophages and ILC2.

In response to diverse stimuli, macrophages may undergo classical M1 activation (stimulated
by TLR ligands or IFN-?) or alternative M2 polarization (stimulated by IL-4/IL-13
axis). M2-macrophages, unlike M1-macrophages, have a low expression of IL-12, high
expression of IL-10, TGF-?, and arginase-1, and exhibit anti-inflammatory and immunosuppressive
functions 132]. Thus, by using a mice model with IL-4R?/? macrophages it was shown that generation
of M2-macrophages is essential for mice survival during schistosomiasis through their
inhibitory effects on Th1 response 133]. Further, Litomosoides sigmodontis (L. sigmodontis) infection in mice recruited a F4/80+ population of alternatively activated macrophages
that potently inhibited Ag-specific CD4+ T-cell proliferation 134]. Moreover, the type-1 cystatin derived from the liver fluke Clonorchis sinensis (C. sinensis) significantly reduced intestinal inflammation by recruiting IL-10-secreting macrophages
in a dextran-sodium-sulfate (DSS)-induced colitis mice model 135].

ILCs are a recently described population of lymphocytes that lack B- and T-cell lineage
markers and are divided into three subtypes: ILC1, which secrete IFN-?; ILC2, which
secrete IL-5 and IL-13; and ILC3, which secrete IL-17 and IL-22. ILCs play a role
in host anti-helminth protective immunity and are able to initiate allergy. The cytokines
IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) have been shown to drive ILC2
expansion 136],137]. The immunomodulatory effects of helminthes on this cell subtype remain unclear.
However, McSorley et al. 138] recently highlighted the potential immunomodulatory effect of helminthes on ILC in
an airway allergy mice model. H. polygyrus ES products were found to suppress ILC2 response by blocking IL-33 secretion, thereby
abolishing the allergic response to allergens. Further studies are needed to clarify
the role of these innate immune cells in the protective effect induced by helminthes.

Microbiota regulation

Apart from its actions on the immune system, helminthes may modulate the bacterial
composition of intestinal flora, promoting the growth of gut microorganisms typically
considered to be ‘probiotics’ 139],140]. As mentioned above, the microbiome plays an essential role in regulating the immune
system 68],69] and disrupting the balance of the gut microbiome may promote the development of autoimmune
diseases 70]-72]. Additionally, as previously discussed, many factors, to date, have been involved
in the modulation of the intestinal microbiota and may in this way prevent the development
of inflammatory disorders. Similarly, by maintaining the microbiome balance, helminthes
likely contribute to the prevention of immune-mediated disorders.

The mechanisms through which some microorganisms can manipulate the host’s immune
response to ensure their own survival are complex and interlinked, and will need to
be specified in further studies. However, it is likely that immunomodulatory strategies
depend on both the pathogen involved and host factors such as genetic background and
the local microenvironment. Meanwhile, the exceptional immunomodulatory properties
of these microorganisms, particularly helminthes, which are the best characterized
to date, led to their successful application in autoimmune disease treatment in both
animal models and clinical trials, thereby providing evidence of the HH and paving
the way to a new therapeutic potential.

Proof of concept of the HH: helminth therapy

The recognition of the extensive immunoregulatory properties of numerous microorganisms
from our environment, especially helminthes, initially suggested in the HH and since
then widely demonstrated, led, in the 1990s, to their therapeutic application in experimental
models and clinical studies of several immune-mediated conditions, including IBD,
MS, RA, T1D, celiac disease, Grave’s disease, and psoriasis. Various species of helminthes
and different approaches were evaluated, including colonization by helminthes larvae,
oral administration of helminthes ova, and the use of helminth-derived antigens. Helminth-derived
therapies, as detailed below, were found to both prevent or delay the onset and reduce
the severity of autoimmune diseases in both animal models and clinical trials. These
experiments are summarized in Table 1.

Table 1. Experimental and clinical studies of helminth-derived therapies in autoimmune diseases

Inflammatory bowel disease (IBD)

Strong experimental data using helminthes in various murine models of colitis have
suggested the benefit of helminth therapy in IBD 122],123],135],141]-148]. Thus S. mansoni soluble egg antigen (SEA) 142] or cercariae 143],145] exposure were shown to significantly attenuate trinitrobenzene sulfonic acid (TNBS)-induced
142],143] or DSS-induced 145] colitis in mice by enhancing IL-4 and IL-10 expression, decreasing INF-? levels 142],143], or through induction of F4/80+ macrophages 145]. Infection of mice with either T. spiralis cercariae 141] or antigens 147] prior to dinitrobenzene sulfonic acid (DNBS)-colitis induction reduced its severity
and was correlated with higher IL-4, IL-13, and TGF-? production and down-regulation
of IFN-?, IL-1?, myeloperoxidase activity, and inducible nitric oxide synthase expression.
Similarly, H polygyrus122],123], Hymenoleptis diminuta (H. diminuta) 144], Schistosoma japonicum (S. japonicum) 146], and C. sinensis135] were found to attenuate or prevent the Rag IL-10?/? T-cell transfer model of colitis
and DNBS-, TNBS-, and DSS-induced colitis, respectively, through mechanisms including
promotion of Treg cells, Th2-cytokines (IL-4, IL-5, IL-10), tolerogenic DCs, and M2-macrophages,
as well as inhibition of IFN-? and IL-17 secretion.

These encouraging results have led to several clinical trials 149]-152] evaluating the safety and therapeutic potential of helminth therapy, mainly Trichuris suis (T. suis) ova (TSO) in IBD patients. T. suis is a pig whipworm able to colonize a human host only for a short period of time.
Following an initial study 149] suggesting that TSO given orally could be a safe and effective treatment of IBD,
Summers et al. conducted two trials; an open-label study in 29 patients suffering
from active CD as defined by a Crohn’s disease activity index (CDAI) ?220 150], and a randomized double blind placebo-controlled trial in 54 patients with active
UC, defined by an ulcerative disease activity index (UCDAI) ?4 151]. All CD patients ingested 2,500 TSO every 3 weeks for 24 weeks; UC received either
2,500 TSO or placebo orally at 2-week intervals for 12 weeks. At the studies’ conclusion,
79.3% of CD patients responded (decrease in CDAI 100 points or CDAI 150) and 72.4%
remitted (CDAI 150), whereas, although UC remission rates between the two groups
were not significantly different, improvement of the UCDAI occurred in 43.3% of patients
with ova treatment compared with 16.7% given placebo. No side effects were reported
in either study. Although T. suis immunoregulatory mechanisms were not studied in these trials, it is assumed that
this involves the modulation of Th1, Th2, Treg, and Th17 subsets as suggested in murine
models. In October 2013, the TRUST-I phase 2 clinical trial 153] evaluating TSO treatment (7,500 ova every 2 weeks for 12 weeks) versus placebo in
250 moderate-to-severe CD patients failed to reach its primary (100-point CDAI decrease)
and key secondary (achieving CDAI 150) endpoints. Despite these discouraging results,
the authors assumed, according to subgroup analyses, that the effectiveness of TSO
could be higher in severe patients (CDAI 290). A number of clinical trials using
TSO in IBD patients have been performed (listed in 95],154]), and the human hookworm Necator americanus (N. americanus) has been suggested as an alternative to T. suis since it is easier to use due to longer-lasting effects 155] and appears to be well tolerated 156].

Multiple sclerosis (MS)

Many studies 104],106],121],129],157]-163] conducted in EAE mice, the main animal model of MS, have highlighted the interest
of helminth-derived therapies in this disease. Indeed, prior treatment of mice before
EAE induction with either S. mansoni ova 160], cercariae 158], or antigen 162] significantly reduced the incidence as well as the severity of EAE as measured by
clinical scores and central nervous system (CNS) inflammation. This protective effect
was associated with decreased IL-12, IFN-?, and TNF-? secretion and higher IL-4, IL-10,
and TGF-? levels in periphery. It is of interest to note that increased IL-4-secreting
neuroantigen-specific T-cells and reduced macrophage and CD4+ T-cell infiltration
were observed in the CNS of infected mice as compared to controls. Similarly, pretreatment
of EAE Dark Agouti rats with T. spiralis ES products (ES-L1) 104],163] ameliorated the clinical and histological severity of induced EAE in a dose-dependent
manner. The mechanisms involved an inhibition of Th1 and Th17 cytokines (IFN-?, IL-17)
and a promotion of Th2 cytokines (IL-4, IL-10) and TGF-?, as well as induction of
Treg cells. Transfer of splenic T-cells from T. spiralis-infected rats into EAE rats led to protection from disease development in some cases.
As indicated above, injection of DCs stimulated with T. spiralis ES-L1 products 7 days before EAE induction 121] was also found to ameliorate EAE by increasing IL-4, IL-10, TGF-?, and Treg levels
and decreasing IFN-? and IL-17 secretion, both at the systemic level and in target
organs. Further, several studies have demonstrated the benefit of H. polygyrus129], Trichinella pseudospiralis106], S. japonicum164], Fasciola hepatica161], and Taenia crassiceps159] infections in preventing or delaying the onset of EAE and improving its severity.
These effects result from inhibition of Th1- and Th17-responses with lower IFN-?,
TNF-?, IL-6, and IL-17 secretion, reduction of CNS inflammatory infiltrates, enhanced
Th2 cytokine production (IL-4, IL-10), and proliferation of Breg, Treg, M2-macrophage,
and tolerogenic DC populations.

To date, few studies have evaluated the therapeutic potential of helminthes in MS
patients. The most noteworthy observations were reported by Correale et al. 131],165],166] through several prospective studies comparing a series of 12 patients with MS naturally
infected with different species of helminthes (Hymenoleptis nanan, Trichuris trichura, Ascaris lumbricoides, Strongyloides stercolaris, and Enterobius vermicularis) with 12 non-infected MS patients as well as with infected patients without MS and
healthy subjects. The authors assessed the clinical, radiological, and immunological
characteristics of each group of patients with a 4.6-year follow-up extended to 7.5 years
in a later report. During the initial 4.6-year follow-up period 165], parasite-infected MS patients showed a significantly lower number of relapses, reduced
disability scores, and lower magnetic resonance imaging activity compared to uninfected
MS subjects. Infected patients showed higher IL-10- and TGF-?- and lower IL-12- and
IFN-?-secreting cell levels. Moreover, Treg and IL-10-secreting Breg cells were significantly
increased in parasite-infected patients compared to other groups 131]. Interestingly, these Breg cells were also found to produce greater amounts of brain-derived
neurotrophic factor and nerve growth factor, raising the possibility that these cells
may exert a neuroprotective effect on the CNS 131]. Anti-helminthic treatment of 4 of the 12 patients treated due to gastrointestinal
symptoms 166] led to a significant rise in clinical and radiological MS activities and in the number
of IFN-?- and IL-12-secreting cells together with a fall in the levels of Treg cells
and TGF-?- and IL-10-secreting cells, which became evident 3 months after anti-helminthic
treatment began.

A safety phase 1 study (phase 1 Helminthes-induced Immunomodulatory Therapy – HINT-1)
167] inoculating 2,500 TSO orally every 2 weeks for 3 months in five relapsing-remitting
MS patients reported mild gastrointestinal side effects. The mean number of new MRIlesions
fell from 6.6 at baseline to 2.0 after 3 months of TSO administration and rose again
to 5.8 at 2 months after the end of the study. In view of these encouraging results,
several clinical trials are already underway or planned, including an extension of
HINT-1 as well as numerous studies evaluating TSO or dermally-administrated hookworm
N. americanus as therapies in MS patients (listed in 95],154],168]).

Rheumatoid arthritis (RA)

Several helminthic products with immunomodulatory properties have been studied in
mice models of RA, the filarial-derived glycoprotein ES-62 being the best characterized
169]. However, so far, no helminth-derived molecules have been used in RA clinical trials.

ES-62 is a tetrameric phosphorylcholine (PC)-containing glycoprotein secreted by the
rodent filarial nematode A. vitae first described in 1989 170]. A decade ago, this glycoprotein was demonstrated to significantly reduce the initiation,
severity, and progression of CIA in DBA/1 mice, a murine model of RA 114]. Since then, its protective mechanisms in CIA mice have been elucidated by Harnett
et al. through several experiments 108],128],171]-173]. ES-62 inhibits Th1- and Th17-responses resulting in decreased levels of IFN-?, TNF-?,
IL-6, and IL-17 both in draining lymph nodes and joints of CIA animals. Regarding
Th-17 response, ES-62 down-regulates IL-17 secretion by both innate (?? T cells) and
adaptive (Th17 CD4+ cells) cells via DC-dependent and independent mechanisms 108]. It decreases collagen-specific IgG2a antibody production and reduces effector B-cells,
particularly plasma cell activation, proliferation, and joint infiltration. Conversely,
ES-62 was found to up-regulate IL-10 secretion by splenocytes and to restore IL-10-secreting
Breg cell levels in CIA mice 128]. IL-22 plays a dual role in CIA, being pathogenic during the initiation phase while
acting to resolve inflammation and joint damage during established disease. Exposure
to ES-62 in vivo suppressed the early peak of IL-22 but induced strong expression at later time points,
serum levels of IL-22 correlating inversely with articular scores 172]. Finally, ES-62 is also able to modulate DCs to promote a Th2 response. Interestingly,
most of the anti-inflammatory actions of ES-62 in CIA have been shown to be related
to its PC moiety 171]. Indeed, the whole ES-62 molecule and a PC-ovalbumin conjugate successfully reduced
disease severity by suppressing Th1 cytokine production while a PC-free recombinant
form of ES-62 failed to prevent CIA progression 173]. Furthermore, a sulfone-containing PC analogue (11a) was designed and demonstrated
to be effective in protecting DBA/1 mice from developing CIA 173]. Apart from A. vitae secretory product, several helminthes, including S. mansoni107], H. diminuta174], F. hepatica175], S. japonicum176], H. polygyrus, and Nippostrongylus brasiliensis177], have been demonstrated to effectively prevent RA-like disease in mice models through
inhibition of Th1/Th17 cytokine secretion, induction of tolerogenic DCs, and promotion
of Treg cell proliferation.

Type-1 diabetes (T1D)

No clinical trial using helminth-derived therapies has so far been conducted in T1D
patients. However, a large body of experimental data from non-obese diabetic (NOD)
mice 109]-113],115],178]-181], the murine model of T1D, suggest their effectiveness, especially for S. mansoni111]-113],178],179].

Cooke et al. 178] were the first to suggest S. mansoni infection as a preventive treatment in NOD mice. Infection with S. mansoni cercariae or SEA significantly reduced the spontaneous incidence of diabetes and
prevented the class switch from IgM to IgG anti-insulin autoantibodies normally seen
in most NOD mice as they approach overt diabetes. The authors subsequently reproduced
this protective effect in a series of experiments by specifying its mechanisms 111]-113],179]. S. mansoni SEA completely prevented the occurrence of T1D when injected to 4-week-old NOD mice
111] by expanding Treg cells in a TGF-?-dependent manner and Th2 cells with increased
secretion of IL-4, IL-5, IL-10, and IL-13. Moreover, T-cells from SEA-treated mice
exhibited a reduced ability to transfer diabetes to NOD-severe combined immunodeficiency
recipients. NOD mice are known to have deficiency in V alpha 14i NKT cells, the expansion
of this population preventing diabetes onset 182]. S. mansoni SEA increased the number of V alpha 14i NKT cells and induced functional changes
in DCs, found to secrete more IL-10 and less IL-12. Recently, ?-1, one of the two
major glycoproteins secreted by S. mansoni ova, was demonstrated to be responsible for its effects 113]. Several studies also evidenced the efficacy of H. polygyrus109],180], T. spiralis109], L. sigmodontis110],115], and Dirofilaria immitis (D. immitis) recombinant antigen 181] to completely prevent the occurrence of diabetes in NOD mice especially by eliciting
a Th2-type response and promoting the proliferation of Treg cells, thereby markedly
inhibiting pancreatic insulitis.

Other immune-mediated diseases

Experimental and clinical data evaluating helminth therapy in other inflammatory conditions
are scarce.

Regarding celiac disease, a double-blinded placebo-controlled study 183] explored the effects of N. americanus cutaneous inoculations in 10 patients compared with 10 non-infected patients. Inoculations
of 15 third-stage larvae were performed at weeks 0 and 12, and a 5-day oral gluten
challenge was undertaken at week 20. No significant reduction in symptom severity
was seen in infected subjects compared to non-infected patients. However, immunological
data from the clinical trial analyzed in a subsequent study 184] found that basal Th1- and Th17-responses were inhibited in the duodenum of hookworm-infected
patients with decreased IFN-? and IL-17A secretion. The authors hypothesized that
the infective dose of hookworms used in the trial may be insufficient to effectively
suppress the immunopathology of celiac disease.

In SLE, a recent study 185] analyzed, for the first time, the effects of infection of MRL/lpr lupus mice with
the trematode S. mansoni. The infection completely changed the phenotype of glomerulonephritis in MRL/lpr mice,
switching from a severe diffuse proliferative Th1-mediated pattern towards a membranous
Th2-mediated nephritis associated with a better prognosis. This effect was associated
with a modulation of the cytokine profile shifting from a Th1 to a Th2 polarization
with increased rates of IL-4, IL-5, IL-10, and TGF-?.

In Grave’s disease, prophylactic use of S. mansoni product homogenates in a mouse model prior to disease induction prevented its development
through Th2 polarization 186]. This was associated with decreased IgG2a subclass anti-thyroid stimulating hormone
receptor antibody production, lower IFN-? levels, and enhanced Treg proliferation.

Finally, one experiment 187] conducted on the fsn/fsn mouse model of psoriasis demonstrated the efficacy of subcutaneous
S. mansoni LNFPIII glycan treatment to prevent the appearance of psoriatic skin lesions as compared
with control mice. Skin cells from LNFPIII-treated mice secreted lower amounts of
IFN-? and increased levels of IL-13, evidencing a shift toward a Th2-type response.
It is noted that several psoriasis clinical trials using TSO are planned 95],154].

Future perspectives and challenges

The emerging potential of helminth-derived therapy in autoimmune diseases is raising
growing interest. The encouraging data from mouse models and early clinical studies
have led to an increase in the number of phase 1 and 2 clinical trial projects in
IBD, MS, RA, psoriasis, and celiac disease 154]. Recently, we have successfully employed helminthes’ PC derivatives to treat colitis
using a prophylactic protocol in mice 188]. Nevertheless, the field of inflammatory Th1/Th17-mediated diseases that may benefit
from such treatment is wide and studies of helminth-derived therapy have only just
commenced. Much work remains to be done, including analyzing and extracting the molecules
responsible for helminth regulating properties, clarifying their action on the immune
system, confirming previous findings in larger prospective trials, and identifying
other diseases eligible for this new type of immunomodulation. Furthermore, these
immunotherapies require the greatest caution, especially regarding the unclear long-term
effects of helminth immunomodulation. The manipulation of the immune response could
lead to compromise in the defense mechanisms against other pathogens or cancers. The
possibility of parasites inducing chronic infection, which may be less controllable,
also needs to be considered. Therefore, the use of helminth-derived molecules appears
to be a more attractive and safe solution 95],154]. If handled cautiously, helminthes might become part of our future therapeutic armamentarium.