The Hygiene Hypothesis and New Perspectives—Current Challenges Meeting an Old Postulate

The Hygiene Hypothesis and New

Perspectives—Current Challenges

Meeting an Old Postulate

Holger Garn

1,2

, Daniel Piotr Potaczek

and Petra Ina Pfefferle

2,3,4

Translational Inﬂammation Research Division & Core Facility for Single Cell Multiomics, Medical Faculty, Biochemical

Pharmacological Center (BPC), Philipps University of Marburg, Marburg, Germany,

German Center for Lung Research

(DZL), Marburg, Germany,

Comprehensive Biobank Marburg (CBBMR), Medical Faculty, Philipps University of Marburg,

Marburg, Germany,

German Biobank Alliance (GBA), Marburg, Germany

During its 30 years history, the Hygiene Hypothesis has shown itself to be adaptable

whenever it has been challenged by new scientiﬁc developments and this is a still a

continuously ongoing process. In this regard, the mini review aims to discuss some

selected new developments in relation to their impact on further ﬁne-tuning and expansion

of the Hygiene Hypothesis. This will include the role of recently discovered classes of

innate and adaptive immune cells that challenges the old Th1/Th2 paradigm, the

applicability of the Hygiene Hypothesis to newly identiﬁed allergy/asthma phenotypes

with diverse underlying pathomechanistic endotype s, and the increasing knowledge

derived from epig enetic studies that leads t o better unde rstanding of mechanisms

involved in the translation of environmental impacts on biological systems. Further, we

discuss in brief the expansion of the Hygiene Hypothesis to other disease areas like

psychiatric disorders and cancer and conclude that the continuously developing Hygiene

Hypothesis may provide a more generalized explanation for health burden in highly

industrialized countries also relation to global changes.

Keywords: hygiene hypothesis, allergy, asthma, non-communicable inﬂammatory diseases, chronic inﬂammation

INTRODUCTION

Throughout its history, the Hygiene Hypothesis has shown itself to be adaptable and ﬂexible

whenever it has been challenged by innovation in science (1). A number of new ﬁndings need to be

considered in this ongoing revisiting process: The originally proposed Th1/Th2 paradigm is

challenged by currently elucidated new classes of effector and regulating immune cells pointing

out to a more complex immune network involved in allergy development (2). Studies on biomarkers

and deep phenotyping techniques changed our understanding of asthma as a uniform disease in

favor of distinct phenotypes that are driven by different causations (3). The emerging ﬁeld of

epigenetics enables us to ﬁll the black box of “gene-by-environment interactions” with conveying

mechanisms ( 4). Currently recognized epigenetic pathways overlapping between chronic

inﬂammatory diseases and other disorders such as psychiatric conditions or cancer might extend

the Hygiene Hypothesis toward a model explaining in a broader sense the rise of health burdens in

westernized societies (5). Finally, the world-wide challenge caused by the climate changes will not

Frontiers in Immunology | www.frontiersin.org March 2021 | Volume 12 | Article 6370871

Edited by:

Maziar Divangahi,

McGill University, Canada

Reviewed by:

Eva Kaufmann,

Research Institute of the McGill

University Health Center, Canada

James Martin,

McGill University, Canada

*Correspondence:

Holger Garn

garn@staff.uni-marburg.de

Specialty section:

This article was submitted to

Microbial Immunology,

a section of the journal

Frontiers in Immunology

Received: 02 December 2020

Accepted: 04 March 2021

Published: 18 March 2021

Citation:

Garn H, Potaczek DP and Pfefferle PI

(2021) The Hygiene Hypothesis and

New Perspectives—Current

Challenges Meeting an Old Postulate.

Front. Immunol. 12:637087.

doi: 10.3389/fimmu.2021.637087

MINI REVIEW

published: 18 March 2021

doi: 10.3389/fimmu.2021.637087

leave the consequences of Hygiene Hypothesis unaffected.

Changing life-styles are closely related to measures

implemented to slow down CO

emissions and to stabilize the

world climate (6).

CHALLENGES FROM IMMUNOLOGY –

THE IMMUNE SYSTEM BECOMES MORE

COMPLEX

Parallel to the revisions that Hygiene Hypothesis has undergone

over time (7), our perception of the mechanisms underlying

cellular and humoral immune responses has changed

fundamentally over the last decades. High-resolution ﬂow

cytometry and cell sorting and, most recently, single cell

multiomics-based analyses provided a deeper insight into the

phenotypic characterization, function, and development of

diverse classes of hematopoietic cell types. The dichotomous

model of divergent Th1 and Th2 responses was signiﬁcantly

expanded by the discovery that T lymph ocytes represent a

branched network of subsets, characterized by a high level of

plasticity and adaptability (8). Namely, Sakaguchi’s discovery of

regulatory T-cell (Treg) subsets provid ed a signiﬁcant new

impetus to researchers investigating the immunological origin

of allergic and autoimmune diseases and their prevention under

healthy conditions and pointed out new strategies to combat

those maladies (9, 10). Moreover, the discovery of new classes of

effector cells and their cellular interactions added relevant

evidence to the ﬁeld. As one example, innate lymphoid cells

(ILC) became of main interest as they have been shown to be

both directly and indirectly associated with and involved in the

development of allergic responses (11). This unique class of

effector cells lacks a clonally distributed antigen receptor which

thus resemble innate immune cells characterized by (antigen)

unspeciﬁc activation, however, they exert T helper (Th)-like

effector cell activities (12). According to their expression of

effector cytokines and transcription factors ILC have been

classiﬁed into three groups: ILC1, ILC2, and ILC3 (13). While

ILC1 produce interfer on-gamma (IFNg) and tumor necrosis

factor a (TNFa) and, similarly to Th1 cells, express T-bet,

ILC2 are able to produce Th2 cytokines such as IL-5 and IL-

13, like Th2 cells under the control of the transcription factor

GATA-3. ILC3 are similar to Th17 cells and release IL-17A and

IL-22 as well as granulocyte macrophage colony stimulating

factor (GM-CSF). In animal models of allergic airway

inﬂammation as well as in human allergic asthmatics ILC2 are

present at elevated frequencies within the lung and airways

epithelial compartments where they were found to produce

high amounts of the type-2 cytokines IL-5 and IL-13 (14).

Within the last years, ILC2 have been recognized as early

promoters to establish and maintain allergic airway

inﬂamm atory res ponses but also as protectors promoting

repair processes of the lung epithelium (15, 16).

A potential link between the Hygiene Hypothesis and the

function of ILC lineages comes from the gut. The symbiotic

interaction between immune cells and the microbiota in the gut

is principally decisive for the development o f tolerance or

pathogenicity. The ILC3 lineage is essential in the development

of lymphoid follicles and Peyer’s patches in the gut and was

shown to be crucial for the maintenance of a well-balanced

symbiosis with the microbiota (17). The host microbiota itself

might play an important role in determining ILC subsets

speciﬁcity as indicated by results coming from experimental

approaches. Sepahi et al. very recently reported that short

chain fatty acids (SCFA) arising from dietary ﬁbers by

microbial fermentation in the intestine induced expansion of

prevailing ILC subsets. By triggering ILC subset expansion via G-

protein-coupled receptors (GPCR) those dietary metabolites

contribute to the homeostasis in the local compartment (18).

Another mechanism to induce repair and homeostatic

conditions at epithelial surfaces is mediated via IL-22-

producing ILC3 in response to the microbiota. In interaction

with IL-18 produced by the epithelial cells, IL-22 is involved in

the promotion of repair and remodeling processes as well as in

the maintenance of the gut homeostasis (19

). By acting as

mediators between the microbiota and the host ILC are

recognized as crucial in the early host response to

microbial stimuli.

CHALLENGES FROM CHANGING

ENVIRONMENTS – CAN EPIGENETICS

PROVIDE THE MISSING LINK TO EXPLAIN

“GENE-BY-ENVIRONMENT

INTERACTIONS”?

Very recently, damaging factors that jeopardize the normal

development or disturb the balance of an established immune

system have come into the focus of research on allergic diseases.

Environmental changes caused by in- and outdoor pollution (20,

21) and the global warming impact the atopic epidemic and some

attempts were undertaken recently to integrate these scenarios

into the concept of the Hygiene Hypothesis on the basis of

epigenetic changes driven by gene-by-environment

interactions (22).

In contrast to our ancestors who spent most of their life time

outdoors and thus close to a natural environment, post-modern

and mainly urban life-styles are characterized by a signiﬁcantly

higher proportion of indoor activities. These changing habits

underline the potential importance of indoor air composition on

the development of allergic diseases and further emphasize the

role of the environmental microbiota (23). Indoor air in urban

homes is often burdened with elevated levels of molds which are

found to be harmful to the airways and favor the development of

airway inﬂammation and asthma (24). Against the background

of growing climate awareness and the resulting increased efforts

to reduce energy consump tion and CO

emissions, current

research on these indoor exposures in homes with improved

house insulation points out to an up-coming health problem.

Enrichment of volatile organic compounds released from

furniture or brought in by tobacco smoke as well perennial

Garn et al. Hygiene Hypothesis New Perspectives

Frontiers in Immunology | www.frontiersin.org March 2021 | Volume 12 | Article 6370872

allergens and molds will jeopardize mainly infants as the

developing immune system and the growing lung are highly

susceptible to these damage factors (25). Already the fetus might

become affected by these components (26). This was exemplarily

shown for tobacco smoke in a transgenerational case control

study conducted to assess the risk for asthma by prenatal

smoking. Grandmothers and mothers of asthmatic and non-

asthmatic children were asked about smoking habits during their

own pregnancy. The study reported an odds ratio twice as high

for children to develop asthma in families where grandmothers

frequently smoked during the mother’s fetal period (27).

At that point the Hygiene Hypothesis was in line with an

upcoming general idea that non-inherited/non communicable

diseases like allergies and asthma develop on the background of

an inappropriate interaction between environmental exposures

and a given genotype to shape a speciﬁc (disease) phenotype.

Though based on the concept of a so-called epigenetic landscape

post ulat ed by Waddington already in the 50ties of the last

century, the underlying molecular mechanisms of epigenetic

programming had still been the “missing link” in the scenario

of gene-by-environment-interactions (28). By discovering

mechanisms such as DNA methylation, diverse histone

modiﬁcations and microRNA regulation as molecular

mechanisms underlying epigenetic regulation of gene

expression, an exciting new ﬁeld of research was opened that

currently has a strong impact on research aiming to unravel the

still existing mysteries of allergy development and prevention

(29–31).

Indeed, epigenetic mechanisms have meanwhile clearly been

demonstrated to be involved in mediating the effects of

environmental factors increasing or decreasing the risk of

allergy development (4). Pro-allergic environmental inﬂuences

can be exempliﬁed by pollution. For instance, higher in utero

exposure to polycyclic aromatic hydrocarbons (PAH) has been

shown to be associated with increased cord blood leukocyte

DNA methylation at the promoter of the IFNg-encoding gene

(32, 33). Moreover, in Treg isolate d from periphe ral blood

mononuclear cells, higher PAH exposure has been correlated

with elevated DNA methylation at the promoter of the gene

encoding FOXP3, a master regulator of Treg development and

activities, with the effect being stronger in asthmatic than in non-

asthmatic children (34).

After epidemiological studies had demonstrated an

association between spending early life time in speciﬁc

agricultural environments and protection against the

development of allergies in childhood (35, 36), functional

investigations of various types started to clarify which elements

of farming, such as contact with farm animals, consumption of

raw cow’s milk, exposure to so-called farm-dust, and others,

mechanistically underlie this observation. DNA demethylation at

the FOXP3-encoding locus related to higher expression of the

gene and activation of Treg (37) has been associated in cord

blood with maternal consumption of raw cow’s milk (38) and in

children’s whole blood with early-life ingestion of raw cow’s milk

(39). Compared to processed shop milk, pretreatment with raw

cow’s milk reduced features of the disease in mice subjected to a

model of food allergy and this effect was mediated by changes in

histone acetylation patterns at crucial T cell-related genes (40

41). Interestingly enough, unprocessed cow’smilkhasbeen

shown to contain miRNAs potentially affecting the expression

of important allergy-related immu ne genes, which might

contribute to its protective effects against asthma (42). Several

bacteria have been isolated from the farming environment, for

instance Acinetobacter lwofﬁi (A. lwofﬁi), which were

demonstrated to diminish the development of allergic

symptoms in mu rine models (43). A. lwofﬁi-mediated

protection against allergic airway inﬂammation has been

observed in mouse models also transmaternally and shown to

be IFNg− dependent, with this effect being at least partly

mediated by preservation of histone H4 acetylation at the

promoter of the IFNg-encoding gene as observed in CD4

cells isolated from spleens of the offspring (44, 45).

CHALLENGES FROM THE CLINICS AND

LESSONS FROM ANIMAL MODELS –

ASTHMA PHENOTYPES AND THE

HYGIENE HYPOTHESIS

A recurrent debate ﬂared up in the ﬁeld of asthma research

excellently summarized at the time being in a review by Wenzel

in 2012 (46). Coming from clinical heterogeneity of asthma

patients she highlighted that basic inﬂammation patterns differ

in asthma patients which in turn determines the success of the

applied therapeutic strategy. As an early diagnosis and adequate

treatment may prevent the dev elopment of a severe asthma

phenotype later on, novel strategies to discriminate children at

risk from those who will not develop asthma are required (47).

Following the clinical deﬁnition of a phenotype as a result of an

interaction between a given genotype and the environment

Wenzel and colleagues expressed the strong medical need for

novel mole cular and geneti c bi omarkers indicative for the

characterization of such phenotypes and deﬁning the speciﬁc

requirements for stratiﬁed therapies. Based on differences

between Th2-driven atopic asthma and non-atopic asthma a

number of subtypes were deﬁned that evolve and differ with age

and respond differentially to standard drug treatment regimes. It

quickly became clear that the search for a speciﬁc biomarker that

clearly identiﬁes a respective phenotype would not be successful.

Rather, the synopsis of all data collected from a subject known as

“deep phenoty ping” may lead to better understanding of

complex asthmatic conditions (48). Deep phenotyping in the

era of OMICS goes along with a tremendous increase in data that

needs to be analyzed. To handle these big data-sets new

approaches become increasingly employed involving models of

statistical data dimension reduction and machine-learning

strategies (49, 50). The idea behind these data-driven

approaches is to mine data collections and classify them based

on so far hidden patterns behind the data. The hypothesis-free

latent class analysis (LCA) approach represent one of the most

promising tools to identify new or verify proposed asthma (and

Garn et al. Hygiene Hypothesis New Perspectives

Frontiers in Immunology | www.frontiersin.org March 2021 | Volume 12 | Article 6370873

other allergic disease) phenotypes. A ﬁrst LCA approach was

carried out in two cohorts of adult asthmatics. Based on clinical

and personal characteristics Siroux et al. described two distinct

phenotypes in two independent cohorts, a severe phenotype in

which asthma is already established in childhood and a second

type that starts in adulthood with milder outcomes (51). In line

with the Hygiene Hypothesis, these results pointed out speciﬁc

preconditions in infant age which pave the pathway to severe

asthma later in life. LCA analyses in children substantiated the

link between early onset and later disease since early clinical

signs such as current unremitting wheezing episodes are ascribed

to indicate a higher risk for asthma development later in life

while transient wheezing seems to have no pathological

consequences (52).

LCA approaches using data from patient studies elucidated

that there might be phenotypic asthmatic manifestations that

could be explained by the Hygiene Hypothesis while other

phenotypes that might have different pathomechanistic origins

failed to be covered by this supposition (53). Among others, this

discrepancy led to new approaches in pre-clinical animal-based

experimental set-ups as well as investigations based on human

data. New animal models were employed to prove the postulate of

such phenotypes that can be discriminated on the immunological

and histological levels. By switching from the well-established

Ovalbumin (OVA) model, where the sensitization was mainly

achieved by a rather artiﬁcial intraperitoneal allergen sensitization

in the presence of the type-2 driving adjuvant alum, to a more

ﬂexible administration of standardized house dust mite extracts

(HDM) via the nasal route, it was feasible to induce a more

natural and broader spectrum of inﬂammatory phenotypes

ranging from typical allergic eosinophil-dominated respiratory

inﬂammation to airway inﬂammatory conditions almost

exclusively dominated by the inﬂux of neutrophils (54–56).

Such more ﬂexible model systems allow deeper and more

precise investigations of the mechanisms underlying the

development of different pheno types and a much bett er

characterization of the orchestration of different regulatory and

effector T cell subsets in dependence of allergen administration on

a continuum between Th2 and Th1/Th17-driven inﬂammation.

In addition, these mouse models mimic the natural situation

more closely by using common allergens and a potential natural

route of sensitization and thus became helpful for understanding

the diverse clinical phenotypes of allergic and non-allergic as well

as mild and severe asthma (57, 58). B y switching between

different effector T cell responses in these experimental set-ups

substantial knowledge is currently added to our understanding of

clinical manifestations in asthma. In combination with LCA

helping to elucidate clinical phenotypes these recent research

developments strongly boosted a better discrimination between

transient and persistent pediatric allergic conditions as well as

allergic and non-allergic asthma later in life. This new evidence

might lead us to the current limits of the Hygiene Hypothesis.

While IgE-driven allergic asthma undoubtedly ﬁts to the Hygiene

Hypothesis, it is still unclear whether this holds true also for non-

atopic asthma phenotypes the development of which is much

more strongly determined by factors different from a missing

(microbial) education of the immune system. Thus and to further

ﬁne-tune the Hygiene Hypothesis, continuous efforts are required

to distinguish between environmental conditions (such as early

life infection with pathogenic viruses) that are either associated

with the induction of a disease phenotype and/or just contribute

to a shift between distinct inﬂammatory manifestations of allergic

disease phenotypes (59, 60) and those that really result in a

general or a phenotype/endotype-speciﬁc prevention of disease in

line with the Hygiene Hypothesis (43, 61).

CHALLENGES FROM A VIEW OVER THE

FENCE – THE HYGIENE HYPOTHESIS IN

PSYCHIATRIC DISORDERS AND CANCER

The French scientist Bach was the ﬁrst who made the principal

observation of a general inverse correlation in the prevalences of

infectious versus non-communicable chronic in ﬂam matory

diseases within the last seven decades (62). Meanwhil e we

know that abundant exposure to a high diversity of infectious

or even harmless microbes resulting in repeated, low-grade acute

inﬂammatory episodes in early life, associates with lower

prevalence of chronic inﬂammatory disorders accompanied by

low levels of inﬂammatory markers in adulthood. Conversely,

high levels of hygiene during perinatal and early chil dhood

developmental periods characteristic for W estern countries

corresponds to higher levels of inﬂammatory markers

correlating with a higher prevalence of chronic inﬂammatory

disorders later in life. Based on these facts, it has been

hypothesized that frequent episodes of low-grade, in most

cases clinically symptom-free inﬂammation in infancy may

balance responses to inﬂammatory stimuli and thus reduce the

rate of continuation of chronic inﬂammation into adulthood,

most probably by adequately shaping the adaptive immunity-

dependent regulation (23).

Interestingly, this observation considers a broader spectrum

of chronic inﬂammatory conditions beyond allergies that might

ﬁt under the umbrell a of the Hygiene Hypothesis such as

multiple sclerosis, irritable bowel di sease or diabetes type 1

( 63). Moreover, within the last years a similar approach

emerged to explain the tremendou s increase in psychiatric

disorders in westernized countries. Mainly affective disorders

such as major depression and bipolar disorder are increasingly

diagnosed in the western ized world. Patients suffering from

affective and anxiety disorders depict an array of features that

mirror inﬂammatory conditions such as pro-inﬂammatory

cytokines in the blood and the central nervous system

accompanied by elevated levels of circulating C-reactive

prot ein (CRP), activation of lymphocytes and inﬂammatory

cellular signaling pathways (MAPK and NF-kB), w ith the

question of causality remaining a chicken or egg problem (64).

Nevertheless, based on genetic predispositions and epigenetic

modiﬁcations in the brain (nervous system) and the periphery

(immune system), both kinds of pathologies, mood and

inﬂammatory disorders, might become established on the basis

Garn et al. Hygiene Hypothesis New Perspectives

Frontiers in Immunology | www.frontiersin.org March 2021 | Volume 12 | Article 6370874

of a disturbed homeostasis of otherwise tightly balanced adaptive

systems of the body. Interestingly but ﬁtting to the hypothesis,

the microbiota of the gut seems to play a critical role also in the

development of psychiatric disorders as shown by recently

conducted studies (65). Based on an interplay between the gut

and the central nervous system, persistent stress and

maltreatment modiﬁes the nervous system and thereby the

endocrine hypothalamic pituitary axis (HPA) which in turn

alters gut microbiota by cortisol release (66). Dysbiosis in the

gut might lead to a compromised cytokine balance in the blood

followed by an activation of the microglia in the brain after

transfer of inﬂammatory mediators/cytokines through the

blood-brain barrier (67). Further, degradation of beneﬁcial

bacteria in the gut microbiota might result in a loss of

microbiota-derived products such as butyrate which dire ctly

results in the downregulation of g-aminobutyrate, serotonin

and dopamine, all factors directly involved in the neurological

regulation circuits and thus in the genesis of neuropsychiatric

disorders when dysregulated (68).

Finally, to add another example to this collection, there is

increasi ng evidence that similar mechanisms as involv ed in the

protection from allergies might also play a role in the prevention of

oncolog ic diseases (69). There is no doubt that preceding infections

with certain pathogens m ay favor initiation a nd further

developmen t of several tumor disease entities. However, a variety

of recent studies also demonstrated positive ef fects of pathogen-

induced “benign” inﬂammatory processes on cancer development,

even though the underlying mechanisms of this dichotomous

inﬂuence of microbial exposure-mediated immune modulation on

carcinogenesis are not well understood so far (70). As one example,

the origins of childhood leukemia have long been discussed in the

context of microbial stimuli in early childhood. Already at the end

of 20

century the question emerged whether early infections in

childhood may act protectively against childhood acute leukemia by

eliminating expanding aberrant leukocyte clones through well-

trained and established immune mechanisms. In concordance

with the Hygiene Hypothesis, Greaves propagated the “Delayed

Infection Hypothesis” as an explanation for the development of

childhood acute (lymphoblastic/myeloid) leukemia (ALL/AML)

that peaks at the age of 2-5 years of life in af ﬂuent countries (71,

72). In his two hit model, Greaves proposed that based on a

prenatally occurred chromosomal translocation or hyperdiploidy

a pre-leukemic clone is already established around birth (ﬁrst hit). A

second hit event beyond the toddler age then leads to gene deletion

or mutation and subsequent transformation to ALL/AML. While

children suffering from infections and/or exposed to a rich

microbial environment early in life might be ready to prevent that

second aberration, predisposed children with an insufﬁciently

educated immune system due to missing “old friends” contact s in

theearlypostnatallifemightnotbeabletoeliminateexpanding

malignant cell clones (72). A number of studies aimed to prove this

hypothes is by exploiting “day care attendance” before the third year

of life as a proxy for infection. This concept is still a matter of debate.

While the vast majority of these studies could add evidence to the

Greaves hypothesis, some well-conducted studies could not support

his assumptions (73, 74). Recently, a meta-analysis investigated the

farm effect with regard to childhood leukemia and conﬁrmed that

contact to lives tock prov ides pro tect ion not only again st allergi es but

also against childhood leukemia (75). This study might point out to

microbiota as a crucial player in both prevention of allergies and

childhood cancer.

The challenges outlined in this mini review are intended to

stimulate further exciting debates that might result in continuing

revisions and adaptations of the Hygiene Hypothesis. We are aware

that the examples reported in this review may only describe a

limite d subjective selection of the scientiﬁc topics currently

discussed in context of the Hygiene Hypothesis. However, it is

common to all topics that the explanations to unravel the

underlying mechanisms refer to the close and beneﬁcial

relationship between man and microbes as established on the

mucosal surfaces of our body. These interactions result in

adequate shaping of adaptive systems of the body (mainly the

immune system) that enables the whole organisms to appropriately

handle diverse adverse inﬂuences. Without exaggeration, this

ﬁnding might be considered one of the most fundamental

insights of the life sciences within the last thirty years.

AUTHOR CONTRIBUTIONS

All authors contributed by writing and editing of the manuscript.

All authors contributed to the article and approved the

submitted version.

FUNDING

Funded by the German Center for Lung Research (DZL). Open

Access was funded by the Library of the Philipps University

Marburg, Germany.

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Conﬂict of Interest: The authors declare that the research was conducted in the

absence of any commercial or ﬁnancial relationships that could be construed as a

potential conﬂict of interest.

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