Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children

Abstract

Background:
The Amish and Hutterites are U.S. agricultural populations whose lifestyles are remarkably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. The populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities.

Methods:
We studied environmental exposures, genetic ancestry, and immune profiles among 60 Amish and Hutterite children, measuring levels of allergens and endotoxins and assessing the microbiome composition of indoor dust samples. Whole blood was collected to measure serum IgE levels, cytokine responses, and gene expression, and peripheral-blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts obtained from Amish and Hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma.

Results:
Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children, the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high. Differences in microbial composition were also observed in dust samples from Amish and Hutterite homes. Profound differences in the proportions, phenotypes, and functions of innate immune cells were also found between the two groups of children. In a mouse model of experimental allergic asthma, the intranasal instillation of dust extracts from Amish but not Hutterite homes significantly inhibited airway hyperreactivity and eosinophilia. These protective effects were abrogated in mice that were deficient in MyD88 and Trif, molecules that are critical in innate immune signaling.

Conclusions:
The results of our studies in humans and mice indicate that the Amish environment provides protection against asthma by engaging and shaping the innate immune response. (Funded by the National Institutes of Health and others.).

Full text

The new england
journal of medicine
n engl j med 375;5 nejm.org August 4, 2016
411
established in 1812
August 4, 2016
vol. 375 no. 5
The authors’ affiliations are listed in the
Appendix. Address reprint requests to Dr.
Vercelli at the Arizona Respiratory Cen-
ter, University of Arizona, The Bio5 Insti-
tute, Rm. 339, 1657 E. Helen St., Tucson, AZ
85721, or at donata@ email . arizona . edu;
to Dr. Ober at the Department of Human
Genetics, Universit y of Chicago, 920 E.
58th St., CLSC 425, Chicago, IL 60637, or
at c-ober@ genetics . uchicago . edu; or to
Dr. Sperling at the Department of Medi-
cine, University of Chicago, 924 E. 57th
St., JFK R316, Chicago, IL 60637, or at
asperlin@ uchicago . edu.
Ms. Stein, Dr. Hrusch, and Ms. Gozdz
and Drs. von Mutius, Vercelli, Ober, and
Sperling contributed equally to this ar ticle.
N Engl J Me d 2016;375:411-21.
DOI: 10.1056/NEJMo a1508749
Copyright © 2016 Massachusetts Medical Society.
BAC KGRO UND
The Amish and Hutterites are U.S. agricultural populations whose lifest yles are remark
-
ably similar in many respects but whose farming practices, in particular, are distinct;
the former follow traditional farming practices whereas the latter use industrialized
farming practices. The populations also show striking disparities in the prevalence of
asthma, and little is known about the immune responses underlying these disparities.
METHODS
We studied environmental exposures, genetic ancestry, and immune profiles among 60
Amish and Hutterite children, measuring levels of allergens and endotoxins and assess
-
ing the microbiome composition of indoor dust samples. Whole blood was collected to
measure serum IgE levels, cytokine responses, and gene expression, and peripheral-
blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts
obtained from Amish and Hutterite homes on immune and airway responses were
assessed in a murine model of experimental allergic asthma.
RESULTS
Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children,
the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the
Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high.
Differences in microbial composition were also observed in dust samples from Amish
and Hutterite homes. Profound differences in the proportions, phenotypes, and func
-
tions of innate immune cells were also found between the two groups of children. In
a mouse model of experimental allergic asthma, the intranasal instillation of dust ex
-
tracts from Amish but not Hutterite homes signif icantly inhibited airway hyperreactiv
-
ity and eosinophilia. These protective effects were abrogated in mice that were def icient
in MyD88 and Trif, molecules that are critical in innate immune signaling.
CONCLUSIONS
The results of our studies in humans and mice indicate that the Amish environment
provides protection against asthma by engaging and shaping the innate immune re
-
sponse. (Funded by the National Institutes of Health and others.)
abs tr act
Innate Immunity and Asthma Risk in Amish and Hutterite
Farm Children
Michelle M. Stein, B.S., Cara L. Hrusch, Ph.D., Justyna Gozdz, B.A., Catherine Igartua, B.S., Vadim Pivniouk, Ph.D.,
Sean E. Murray, B.S., Julie G. Ledford, Ph.D., Mauricius Marques dos Santos, B.S., Rebecca L. Anderson, M.S.,
Nervana Metwali, Ph.D., Julia W. Neilson, Ph.D., Raina M. Maier, Ph.D., Jack A. Gilbert, Ph.D.,
Mark Holbreich, M.D., Peter S. Thorne, Ph.D., Fernando D. Martinez, M.D., Erika von Mutius, M.D.,
Donata Vercelli, M.D., Carole Ober, Ph.D., and Anne I. Sperling, Ph.D.
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new england journal
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M
any genetic risk factors have
been reported to modify susceptibility
to asthma and allergy,
1,2
but the dra-
matic increase in the prevalence of these condi-
tions in westernized countries in the past half-
century suggests that the environment also plays
a critical role.
3
The importance of environmental
exposures in the development of asthma is most
exquisitely illustrated by epidemiologic studies
conducted in Central Europe that show signif i-
cant protection from asthma and allergic disease
in children raised on traditional dairy farms. In
particular, children’s contact with farm animals
and the associated high microbial exposures
4,5
have been related to the reduced risk.
6,7
However,
the effect of these traditional farming environ-
ments on immune responses is not well defined.
To address this gap in knowledge, we de-
signed a study that compares two distinctive
U.S. farming populations — the Amish of Indi-
ana and the Hutterites of South Dakota — that
recapitulate the differences in the prevalences of
asthma and allerg y observed in farmers and
nonfarmers in Europe. These two particular
groups of farmers originated in Europe — the
Amish in Switzerland and the Hutterites in
South Tyrol — during the Protestant Reformation
and then emigrated to the United States in the
1700s and 1800s, respectively. Both groups have
since remained reproductively isolated.
8,9
Their
lifestyles are similar with respect to most of the
factors known to influence the risk of asthma,
including large sibship size, high rates of child-
hood vaccination, diets rich in fat, salt, and raw
milk, low rates of childhood obesity, long dura-
tions of breast-feeding, minimal exposure to
tobacco smoke and air pollution, and taboos
against indoor pets. However, whereas the
Amish practice traditional farming, live on single-
family dairy farms, and use horses for fieldwork
and transportation, the Hutterites live on large,
highly industrialized, communal farms. Strik-
ingly, the prevalence of asthma in Amish versus
Hutterite schoolchildren is 5.2% versus 21.3%
and the prevalence of allergic sensitization is
7.2% versus 33.3%, as previously reported.
10,11
Methods
Overview
We characterized the immune profiles of Amish
and Hutterite schoolchildren. Furthermore, we
used mouse models of asthma to study the effect
of the environment on airway responses and to
create a mechanistic framework for the interpre-
tation of our observations in humans.
Study Participants and Study Oversight
In November 2012, we studied 30 Amish children
7 to 14 years of age who lived in Indiana, and in
December 2012 we studied 30 Hutterite children
who lived in South Dakota and were matched with
the Amish children for sex and for age within
1 year. (For information on the characteristics of
the children see Table 1, and the Methods section
in the Supplementary Appendix, available with
the full text of this article at NEJM.org). Written
informed consent was obtained from the parents
and written assent was obtained from the chil-
dren. One parent of each child responded to a
questionnaire on asthma symptoms and previous
diagnoses. The study was approved by the insti-
tutional review boards at the University of Chi-
cago and at St. Vincent Hospital in Indianapolis.
Blood-Sample Collection and Analysis
Whole blood was collected in tubes that con-
tained culture medium alone, medium plus 0.1 μg
per milliliter of lipopolysaccharide, or medium
plus 0.4 μg per milliliter of anti-CD3 plus 0.33 μg
per milliliter of anti-CD28 monoclonal antibod-
ies (TruCulture Blood Collection System, Myriad
RBM). After incubation at 37°C for 30 hours,
A Quick Take
is available at
NEJM.org
Characteristic
Amish
(N = 30)
Hutterite
(N = 30)
Age (yr)
Median 11 12
Range 8–14 7–14
Girls (no.) 10 10
Sibships (no.) 15 14
Children with asthma (no.) 0 6
Positivity for allergen-specific IgE (no.)
>0.7 kUA/liter 5 9
>3.5 kUA/liter 2 9
Serum IgE (kU/liter)
Median 21 64
Interquartile range 10–57 15–288
* UA denotes allergen-specific unit.
Table 1. Demographic and Clinical Characteristics of the Study Populations.*
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Immunity and As thma in Amish and Hut terite Children
supernatant and cells were frozen for use in
gene-expression and cytokine studies. Levels of
26 cytokines were measured with the use of the
Milliplex Map Human TH17 Magnetic Bead
Panel (EMD Millipore) or enzyme-linked immuno-
absorbent assay (eBioscience) in the supernatant,
in accordance with standard protocols. Addi-
tional blood was collected to obtain peripheral-
blood leukocytes for f low cytometry and DNA
isolation, and serum was collected for IgE stud-
ies (as described in the Methods section in the
Supplementary Appendix).
Cryopreserved human peripheral-blood leuko-
cytes were incubated for 10 minutes with pooled
human IgG antibodies (FcX, Biolegend) to block
nonspecif ic antibody binding before undergoing
surface staining with f luorescently conjugated
antibodies (see Table S1 in the Supplementary
Appendix) and intracellular staining for FoxP3
(eBioscience). Flow-cytometry data were acquired
on an LSRFortessa cell analyzer (BD Biosciences),
and acquisition data were analyzed with FlowJo
software (Tree Star).
Dust Collection and Extr act Preparation
Electrostatic dust collectors were placed in one
bedroom and the living room in each of 10
Amish and 10 Hutterite homes to collect air-
borne house dust. All 10 Amish homes and 9 of
10 Hutterite homes housed children who partici-
pated in the study. After 1 month, dust was ana-
lyzed for endotoxin and allergen levels, and ex-
tracts were prepared for studies in mice. In
addition, a vacuum was used to collect dust from
the living-room f loor in Amish homes and from
mattresses in Amish and Hutterite homes for
use in microbiome studies (as described in the
Methods section in the Supplementary Appendix).
Aqueous extracts of house dust from Amish and
Hutterite homes were prepared as described in the
Methods section in the Supplementary Appendix.
Genetic S tudies
RNA was extracted from thawed cells with the
use of AllPrep DNA/RNA Mini Kits (Qiagen).
RNA underwent complementary DNA synthesis
and was then hybridized to HumanHT-12 v4
Expression BeadChip arrays (Illumina). A com-
mon set of 118,789 single-nucleotide polymor-
phisms (SNPs) was genotyped or imputed in the
60 children in the study (see Fig. S1 in the Sup-
plementary Appendix).
Mouse Mode ls
We instilled 50 μl of house-dust extract intra-
nasally every 2 to 3 days (for a total of 14 times)
into 7-week old BALB/c mice (Harlan Laborato-
ries), beginning on day 0. The mice had been
sensitized intraperitoneally with 20 μg of oval-
bumin (grade V, Sigma) plus alum (Pierce) on
days 0 and 14 and were challenged intranasally
with 50 μg of ovalbumin on days 28 and 38.
Beginning 5 days before day 0, we also instilled
50 μl of dust extract from Amish homes intra-
nasally every 2 to 3 days (for a total of 14 times)
in 7-week old, C57BL6 wild-type, MyD88-deficient
mice
12
and in mice def icient in both MyD88 and
Trif
13
(Jackson Laboratories). These mice were
sensitized intraperitoneally with 20 μg of oval-
bumin plus alum on days 0 and 14 and chal-
lenged intranasally with 75 μg of ovalbumin on
days 26, 27, and 28.
Statistical Analysis
Statistical analyses were performed with the use
of R or Prism software (GraphPad). Differences
in the distributions of median cytokine levels
between Amish and Hutterite children were de-
termined with use of a Wilcoxon signed-rank
test. We used linear regression to identify differ-
entially expressed genes in the Amish and Hut-
terite untreated samples of peripheral-blood
leukocytes. The methods of Benjamini and Hoch-
berg
14
were used to control the false discovery
rate. For f low cytometric studies and the study
in mice, differences in cell populations and air-
way resistance were assessed with the use of an
unpaired Student’s t-test. Additional details on
sample processing, quality control, and statisti-
cal analysis for all methods described here are
provided in the Methods section in the Supple-
mentary Appendix.
Results
Asthma and Allergic-Sensitization Rates
and Genetic Ance stry
None of the Amish children and six (20%) of the
Hutterite children had asthma, rates similar to
those reported in earlier studies.
10,11
Levels of
total serum IgE and the number of children
whose levels of IgE against common allergens
were high (defined as more than 3.5 kUA [allergen-
specific unit] per liter) were lower in the Amish
group than in the Hutterite group (Table 1, and
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Table S2 in the Supplementary Appendix). No
statistical differences were observed in levels of
serum Ig isotypes other than IgE (Fig. S2 in the
Supplementary Appendix).
To evaluate whether the differences in the
prevalence of asthma and of allergic sensitiza-
tion and differences in IgE level could be attrib-
uted to population history, we assessed ancestry
by conducting principal component analysis and
compared allele frequencies using genomewide
SNPs. These studies revealed remarkable genetic
similarities between the Amish children and the
Hutterite children, as compared with other Euro-
pean populations
15
(Fig. 1A, and Fig. S3 in the
Supplementary Appendix).
Exposures to Allergens, Microbes,
and Microbial Products
Common allergens (from cats, dogs, house-dust
mites, and cockroaches) were detectable in air-
borne dust from 4 of 10 Amish and 1 of 10 Hut-
terite homes (Table S3 in the Supplementar y
Appendix). In contrast, endotoxin levels were
measurable in airborne dust from all 20 homes,
and median levels were strikingly higher (6.8 times
as high) in Amish homes than in Hutterite homes
(4399 endotoxin units [EU] per square meter vs.
648 EU per square meter, P <0.001) (Fig. 1B).
Analysis of a single pooled sample of mattress
dust from each population revealed different
profiles of the relative abundance of bacteria at
the family level (Fig. S4 in the Supplementary
Appendi x).
Composition and Phenotype of Peripheral-
Blood Leukocytes
Peripheral-blood leukocytes from Amish children
had increased proportions of neutrophils, de-
creased proportions of eosinophils, and similar
proportions of monocytes as compared with
samples from Hutterite children (Fig. 2A). Neu-
trophils from Amish children expressed lower
levels of the chemokine receptor CXCR4 and the
adhesion molecules CD11b and CD11c than did
neutrophils from Hutterite children, suggesting
that these cells may have recently emigrated from
the bone marrow (Fig. 2B). Although propor-
tions of monocytes were similar in Amish and
Hutterite children, monocytes from Amish chil-
dren, unlike those from Hutterite children, ex-
hibited a suppressive phenotype characterized
by lower levels of human leukocyte antigen DR
(HLA-DR) and higher levels of the inhibitory
molecule immunoglobulin-like transcript 3
(ILT3)
16,17
(Fig. 2C). In contrast with previous
studies,
18,19
no significant differences in percent-
Figure 1. Ancestries and Environments of Amish and Hutterite Children.
Panel A shows a principal components plot of the first t wo principal com-
ponents (PC 1 and PC 2) of the analysis of 72,034 single-nucleotide poly-
morphisms (SNPs). Amish and Hutterite genotypes were projected onto
the sample space created by Human Genome Diversity Project (HGDP) for
European populations.
15
Panel B shows endotoxin levels in airborne dust
from 10 Amish and 10 Hutterite homes. Box-and-whisker plots show a hor-
izontal line indicating median value, a box representing the interquar tile
range, and whiskers showing the 95% conf idence interval. The P value was
calculated with the use of the Wilcoxon rank-sum test. EU denotes endo-
toxin units.
ASNP Analysis of Genetic Association
PC 2
20
0
−20
−40
−30 −20 −10 0 10 20 30
PC 1
BEndotoxin Levels in Airborne Dust
EU/m2
Amish
Homes
Hutterite
Homes
20,000
10,000
15,000
5,000
0
P<0.001
Amish Hutterite Basque French North Italian
Russian
Russian Caucasus Sardinian Scottish Tuscan
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Immunity and As thma in Amish and Hut terite Children
Figure 2. Proportions of Peripheral-Blood Leukocytes and Cell-Sur face–Marker Phenotypes in Amish and Hutterite Children.
The percentages of total peripheral-blood leukocy tes (Panel A) were determined with f low cytometry for neutrophils (defined as
CD66b+Siglec-8+), eosinophils (defined as CCR3+Siglec-8+), and monocy tes (defined as CD14+CD66b −). Box-and-whisker plots show a
line indicating median value, with the box showing the interquartile range and whiskers showing the 95% confidence interval. Neutro-
phils (Panel B) were characterized according to the surface expression of CXCR4, CD11b, and CD11c (shown here), along with CXCR1
and CXCR2, expressed as mean fluorescence intensity (MFI). The expression of the interleukin-8 coreceptors CXCR1 and CXCR2 was not
significantly different bet ween groups (P = 0.26 and P = 0.91, respectively). Monoc ytes (Panel C) were characterized for the sur face ex-
pression of HLA-DR and immunoglobulin-like transcripts (ILTs), including ILT3 (shown here). There was no significant difference in the
MFI of inhibitory receptors ILT2 and ILT4 between Amish and Hutterite children (P = 0.69 and P = 0.21, respectively; data not shown),
whereas the surface expression of ILT5 was increased on Amish monocytes (P = 0.0 01; data not shown). All P values were calculated with
the use of an unpaired Student’s t-test. Cell proportions and phenotypes after the exclusion of children with asthma or allergic sensitiza-
tion are shown in Table S4 in the Supplementary Appendix.
CD66b+Siglec-8+ (%)
80
60
40
0Amish Hutterite
BCell-Surface Markers on Neutrophils
ACell Proportions of Peripheral-Blood Leukocytes
Neutrophils
P=0.006
CCR3+Siglec-8+ (%)
12
6
9
3
0Amish Hutterite
Eosinophils
P<0.001
CD14+CD66b– (%)
2
3
1
0Amish Hutterite
Monocytes
P=0.28
MFI
600
400
200
0Amish Hutterite
CXCR4
P<0.001
MFI
15,000
9,000
12,000
6,000
3,000
0Amish Hutterite
CD11b
P<0.001
MFI
600
400
300
100
500
200
0Amish Hutterite
CD11c
P=0.004
CCell-Surface Markers on Monocytes
MFI
4000
3000
1000
2000
0Amish Hutterite
HLA-DR
P=0.004
MFI
2000
1500
2500
500
1000
0Amish Hutterite
ILT3
P=0.004
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ages of T regulatory cells (defined as CD3+,
CD4+, FoxP3+, and CD127−) was observed in
Amish and Hutterite children (0.056±0.054% vs.
0.079±0.081% of peripheral-blood leukocytes,
P = 0.29).
Cytokine Responses to Innate and Adaptive
Stimulation
Cytokine levels were measured in supernatants
from peripheral-blood leukocytes that were cul-
tured for 30 hours, with or without innate
stimuli (lipopolysaccharide) or adaptive stimuli
(combined anti-CD3 and anti-CD28 antibodies).
Twenty-three cytokines were detectable in the
supernatants from peripheral-blood leukocytes
treated with lipopolysaccharide. Median levels of
each of these 23 cytokines were lower in the
Amish children than in the Hutterite children,
and these distributions were signif icantly differ-
ent (P <0.001 by Wilcoxon signed-rank test) (see
Tables S5 and S6 in the Supplementary Appen-
dix). Results were similar after the exclusion of
children who were known to have asthma or
allergies (Table S7 in the Supplementary Appen-
dix). In contrast, after adaptive stimulation, the
overall distributions of median cytokine levels
were not significantly different in peripheral-
blood leukocytes from Amish and Hutterite
children (P = 0.08 by Wilcoxon signed-rank test)
(Table S8 in the Supplementary Appendix).
Gene-Expression Profiles
The striking differences in the proportions of
peripheral-blood leukocytes observed in Amish
and Hutterite children were reflected in the gene-
expression profiles of these cells (Fig. S5 in the
Supplementary Appendix). At a false discovery
rate of 1%, 1449 genes were up-regulated in the
peripheral-blood leukocytes of Amish children
(blue points in Fig. 3A) as compared with 1360
genes up-regulated in the cells of Hutterite chil-
dren (red points in Fig. 3A). These differentially
expressed genes were organized into 15 coexpres-
sion modules with the use of the Whole Genome
Co-Expression Network Analysis (Table S9 in the
Supplementary Appendix). To better understand
the biologic relationships within each set of genes
in each module, we used Ingenuity Pathway
Analysis (Qiagen) to construct unsupervised net-
works on the basis of prior knowledge of the
physical and functional connections bet ween the
molecules encoded by the genes. The most sig-
nif icant network (P = 1.0×10
−30
by Fisher’s exact
test) was in a module that contained 43 genes.
This module was associated with both Amish and
Hutterite stat us (P = 7.1×10
−9
) and was therefore
also associated with the proportions of neutro-
phils (P = 1.5×10
−6
) and eosinophils (P = 1.0×10
−3
).
Eighteen of the genes in this module were over-
expressed in Amish peripheral-blood leukocytes,
and all were clustered in a network that had as
hubs tumor necrosis factor (TNF) and interferon
regulatory factor 7 (IRF7), two key proteins in the
innate immune response to microbial stimuli
(Fig. 3B).
Effects of House-Dus t Extr acts
on Experimental Asthma
To create a framework that would help us to
interpret our observations, which were pointing
toward a protective role of innate immunity, we
used a classic ovalbumin mouse model of aller-
gic asthma, comparing the effects of house dust
obtained from Amish and Hutterite homes by
administering extracts intranasally to mice over
the course of 4 to 5 weeks. Eosinophilia was
observed in bronchoalveolar-lavage samples, and
airway hyperresponsiveness was exacerbated in
mice treated with ovalbumin and Hutterite dust
extracts as compared with mice treated with
ovalbumin alone, findings that were consistent
with the absence of protection from asthma
observed in Hutterite children (Fig. 4A). In con-
trast, inhalation of Amish dust extracts was suf-
ficient to significantly inhibit ovalbumin-induced
airway hyperresponsiveness, eosinophilia in the
bronchoalveolar lavage, and the elevation of se-
rum ovalbumin-specif ic IgE levels (Fig. 4A, and
Table S10 in the Supplementary Appendix). Levels
of lung T regulatory cells (defined as CD3+,
CD4+, and FoxP3+) were not increased (Table S11
in the Supplementary Appendix), and all cyto-
kines measured in bronchoalveolar-lavage sam-
ples, including interleukin-10, were suppressed
in mice that received Amish dust extracts (Table
S12 in the Supplementary Appendix). The inhibi-
tory effects of these extracts in wild-type mice
probably required innate immunity, because
protection was strongly reduced in mice defi-
cient in MyD88 (Fig. 4C) and completely abro-
gated in mice deficient in both MyD88 and Trif
(Fig. 4D), two molecules that are critical to the
development of multiple innate immune-signal-
ing pathways.
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Immunity and As thma in Amish and Hut terite Children
Discussion
Our studies in Amish and Hutterite schoolchil-
dren revealed marked differences in the preva-
lence of asthma despite similar genetic ances-
tries and lifestyles. As compared with the
Hutterites, the Amish, who practice traditional
farming and are exposed to an environment rich
in microbes, showed exceedingly low rates of
asthma and distinct immune profiles that sug-
gest profound effects on innate immunity. Data
generated in an experimental model of asthma
support this notion by showing that the protec-
tive effect of the Amish environment requires
the activation of innate immune signaling.
Analyses of the proportions and gene-expres-
sion profiles of peripheral-blood immune cells in
Amish and Hutterite children revealed differences
in the cells and genes involved in innate immune
responses to microbes. Indeed, neutrophils, eosino-
phils, and monocytes appeared to be major tar-
gets of the distinct environments to which
Amish and Hutterite children are exposed be-
cause these cell types differed between the two
groups in terms of their relative abundance, their
phenotypes, or both. Moreover, the network most
associated with these differences consisted of
innate immune genes. Notable among the genes
that were more highly expressed in the Amish
children was T NFAIP3, which encodes A20, a
ubiquitin-editing enzyme that limits the activity
of multiple inf lammatory pathways that depend
on nuclear factor κB (NF-κB)20 and that has also
been shown to mediate the protective effects of
European farm-dust extracts in murine models
of allergic asthma.21 IRF7, a hub in this network,
Figure 3. Gene -Expression Prof iles in Peripheral-Blood Leukocytes from Amish and Hutterite Children.
In Panel A, a volcano plot shows differences in baseline gene expression in peripheral-blood leukocytes from Amish
and Hutterite children.The x axis indicates the log
2
differences in gene-expression level between groups, with larger
positive values representing genes with higher expression in the Hutterites relative to the Amish (1360 genes, shown
in red points) and larger negative values representing genes with higher expression in the Amish relative to the Hut-
terites (1449 genes, shown in blue points). The y axis shows the –log
10
of the P values for each gene, with larger values
indicating greater statistical significance. The solid horizontal line indicates the 1% false discovery rate. Black points
represent genes from Amish and Hutterite cells for which there was no significant difference in gene expression.
Differences in gene expression remain af ter the data for children with asthma or allergic sensitization were excluded
(Figs. S6 and S7 in the Supplementary Appendix). Changes in gene expression between the two groups after correct-
ing for differences in cell proportion are shown in Figure S4 in the Supplementary Appendix. In Panel B, a network
of differentially expressed genes in untreated peripheral-blood leukocytes is shown. Genes shown in blue have in-
creased expression in Amish children, and the gene shown in red has increased expression in Hutterite children.
The gene shapes indicate the class of each gene’s protein product (spirals denote enzymes, a v-shape denotes cyto-
kines, conjoined circles denote a transcription regulator, hollow upside-down triangles denote kinases, cups denote
transporters, and circles denote other products). Lines represent dif ferent biologic relationships (solid lines indicate
direct interaction, dashed lines indirect interaction, arrows direction of activation, arrows with a horizontal line direc-
tion of activation and inhibition, and lines without arrows binding only).
13601449 STEAP4
ZC3H12A
IRAK3
TRIM25
TRIM8
TNFAIP3
PARP14
TAP2
PARP12
SAMD9L
PSME1
IRF7
DHX58
MAP3K8
STAT2
TNFAIP2
SCO2
RHBDF2
TNF
15
10
5
0
−2 −1 0
Log2 (difference in gene expression)
−Log10 (P value)
1 2
BA
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418
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Figure 4. Ef fects of Amish and Hutterite House-Dust Extracts on Airway Responses in Mouse Models of Allergic Asthma.
Panel A shows the effects of the intranasal instillation of 50 μl of Amish or Hutterite dust extract in 7-week-old mice (BALB/c strain) every 2 to 3 days for a total of 14 times begin-
ning at day 0. The mice were sensitized with ovalbumin (OVA) intraperitoneally on days 0 and 14 and challenged with ovalbumin intranasally on days 28 and 38. Airway resistance
(shown as centimeters of water per milliliter per second and stimulated in response to increasing doses of acet ylcholine administered intravenously) and bronchoalveolar-lavage
(BAL) cellularit y were measured on day 39 (4 to 6 mice per group). The total amount of Amish and Hut terite dust extract administered over the course of the experiment represent-
ed the total load of airborne dust deposited on electrostatic dust collectors placed in Amish or Hutterite homes for 1 month. Statistical differences in experimental measures were
assessed with the use of Student’s t-test. Amish house-dust extrac ts (7.5 mg of dust equivalent in 50 μl) were instilled intranasally every 2 to 3 days for a total of 14 times beginning
5 days before day 0 into 7-week old wild-type mice (Panel B), mice deficient in MyD88 (Panel C), and mice deficient in MyD88 and Trif (Panel D) (all C57BL6 strains). These mice
were sensitized intraperitoneally with 20 μg of ovalbumin on days 0 and 14 and were challenged intranasally with 75 μg of ovalbumin on days 26, 27, and 28. Airway resistance
(shown as an increase from baseline in response to increasing doses of nebulized methacholine) and bronchoalveolar-lavage cellularity were measured on day 30 (12 mice per
group for wild-type mice and 6 mice per group for those def icient in MyD88 or MyD88 and Trif ). Statistical differences in experimental measures were assessed with the use of Stu-
dent’s t-test. I bars represent the standard errors of the data. NS denotes not significant and PBS phosphate-buffered saline.
Saline OVA OVA–Amish Saline OVA OVA–Amish Saline OVA OVA–AmishPBS OVA OVA–
Amish
OVA–
Hutterite
Airway Resistance (cm of water/ml/sec)
20
10
15
5
0
0 1.000.500.25 2.00 4.00
Acetylcholine (µg/g mouse)
BWild Type C57BL6 CC57BL6 MyD88 Knock Out DC57BL6 MyD88–TRif Knock OutABALB/c
P=0.04
P=0.02
P=0.01
P<0.001
P<0.001
P=0.007
Airway Resistance (cm of water/ml/sec)
10
6
8
4
2
0
0 10 30 100
Methacholine (mg/ml)
P=0.03
Airway Resistance (cm of water/ml/sec)
10
6
8
4
2
0
0 10 30 100
Methacholine (mg/ml)
NS
Airway Resistance (cm of water/ml/sec)
10
6
8
4
2
0
0 10 30 100
Methacholine (mg/ml)
NS
BAL Cells (%)
100
50
75
25
BAL Cells (%)
100
50
75
25
BAL Cells (%)
100
50
75
25
BAL Cells (%)
100
50
75
25
0
Eosinophils
Neutrophils
Macrophages
P<0.001
0 0
P=0.02
P=0.01
P<0.001
P=0.008
P<0.001
NS
NS
0
NS
P=0.002
NS
Eosinophils
Neutrophils
Macrophages
Eosinophils
Neutrophils
Macrophages
Eosinophils
Neutrophils
Macrophages
PBS OVA OVA–
Amish
OVA–
Hutterite
Saline OVA OVA–Amish Saline OVA OVA–Amish Saline OVA OVA–Amish
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n engl j med 375;5 nejm.org August 4, 2016
419
Immunity and As thma in Amish and Hut terite Children
regulates type I interferon transcription and is
therefore essential for innate airway responses
against viruses
22
that are linked to susceptibility
to asthma.
23,24
In turn, TR IM8, the one gene in the
network that was more highly expressed in the
Hutterites, acts as a positive regulator of TNF-α–
and interleukin-1β–induced activation of NF-κB.
25
These findings suggest that in the Amish, intense
and presumably sustained exposure to microbes
activates innate pathways that shape and cali-
brate downstream immune responses.
Sustained microbial exposure was also ref lect-
ed in the phenotypes of peripheral innate im-
mune cells in the Amish. Repeated microbial
stimulation can lead to reduced expression of
HLA-DR on monocytes
26,27
and drive immature
neutrophils from the bone marrow.
28-31
Indeed,
Amish children had immature neutrophils bear-
ing markers suggestive of recent emigration
from the bone marrow, and they had monocytes
with reduced expression of HLA-DR and in-
creased expression of ILT3, all of which are sug-
gestive of antiinflammatory function. Proportions
of T regulatory cells and levels of interleukin-10,
which typically mediate immune-balancing effects,
were not increased in the Amish children. How-
ever, qualitative and functional differences in
regulatory-cell populations remain to be defined.
Innate immunity has evolved to sense the
environment and transduce signals that cali-
brate adaptive responses to exogenous antigens.
The proteins MyD88 and Trif are located at the
convergence of multiple innate signaling path-
ways,
32
and deletion of these molecules virtually
disables innate immune responses, thereby also
dysregulating adaptive immunity. The fact that
the loss of protection was more marked in mice
deficient in both MyD88 and Trif than in mice
deficient only in MyD88 points to the involve-
ment of multiple innate pathways. The concor-
dance between findings from studies in humans
and in mice was remarkable: in both studies
protection was accompanied by lower levels of
eosinophils, higher levels of neutrophils, general-
ly suppressed cytokine responses, and no increase
in levels of T regulatory cells or interleukin-10.
Thus, the finding that these features were largely
dependent on innate immune pathways in mice
suggests that innate immune signaling may also
be the primary target of protection in the Amish
children, in whom downstream adaptive immune
responses may also be modulated.
Our study has several limitations. First, we were
unable to include children younger than 6 years
of age, we collected samples at a single time
point, and the numbers of Amish and Hutterite
children in our study were relatively small. As a
result, we may have missed important windows
of immune development or lacked the ability to
detect early, subtle shifts in cell composition,
response, or phenotype that are critical for im-
mune maturation. Second, our microbiome as-
sessments were limited, since only pooled dust
samples from a limited number of homes were
available for the studies in which we assessed
bacterial composition. Therefore, we cannot fur-
ther dissect microbial composition and identif y
potentially protective microbes to target. How-
ever, the striking differences found in endotoxin
levels support the notion that the Amish indoor
environment is much richer in microbial expo-
sures than the Hutterite environment. Third, the
strateg y used for sampling the Hutterite chil-
dren enriched selection for those with asthma,
although the prevalence of asthma in our sample
was similar to that reported in previous popula-
tion-based studies.
11
Moreover, the exclusion of
children with asthma or allergic sensitization
from our analyses of gene expression, cell com-
position, and immune phenotypes did not affect
the outcomes.
Our study in a small number of children was
suff icient to show significant differences in the
prevalence of asthma and in immune profiles,
suggesting that very strong environmental fac-
tors must account for these differences. Indeed,
we showed that there are remarkable genetic
similarities between Amish and Hutterite chil-
dren. Although we interrogated only common
variants, other variants that occur at very low
frequency in these populations are unlikely to
account for the observed large differences in the
prevalence of asthma. In the end, the novelt y of
our work lies in the identif ication of innate im-
munity as the primary target of the protective
Amish environment, a finding supported by re-
sults obtained in both humans and mice. Con-
versely, our work suggests that susceptibility to
asthma may be increased when innate immune
stimulation is weak. A deeper understanding of
the relevant stimuli and the innate immune path-
ways they engage may ultimately pave the way
for the development of effective strategies for
the prevention of asthma.
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n engl j med 375;5 nejm.org August 4, 2016
420
The
new england journal
of
medicine
Supported by the Nat ional I nstit utes of He alth, St. Vincent
Foundat ion, and t he American Academy of Allerg y, Asthma, and
Immunology Foundation.
Disclosure forms provided by the authors are available with
the fu ll text of this art icle at NEJM.org.
We thank t he Hutterite and Amish volunteers and their fam ilies
for participating in this st udy; Gorka Alkor ta-Aranburu, Mait ane
Arrubarrena Orbegozo, Kathleen Bailey, Christine Billstrand, Kelly
Blaine, Daniel Cook, Donna Decker, Moha mmad Jaffer y, Cou rtney
Burrows, Katherine Naught on, Raluca Nicolae, Rob Stanaker,
Meghan Sullivan, and Emma Thompson for assist ance on f ield
trips a nd with sample processing; Peace Ezeh, Amanda Herrell,
Ashley Horner, Kennet h Addison, Dominik Schent en, and Sha ne
Snyder for their contributions to the studies in mice; and Min al
Çalışkan, Yoav Gilad, Jessie Nicodemus-Johnson, Joh n Novembre,
and Matthew St ephens for helpful c omments and statist ical advice.
Appendix
The authors’ affiliations are as follows: the Department of Human Genetics (M.M. Stein, C.I., R.L.A., C.O.), the Department of Medi-
cine, Section of Pulmonary and Critical Care Medicine, and the Committee on Immunology (C.L.H., A.I.S.), the Department of Ecology
and Evolution (J.A.G.), and the Department of Surgery (J.A.G.), University of Chicago, Chicago, and the Institute for Genomic and
Systems Biology, Argonne National Laboratory, Argonne (J.A.G.) — all in Illinois; the NIEHS Training Program in Environmental
Toxicology and Graduate Program in Cellular and Molecular Medicine (J.G.), and the Departments of Cellular and Molecular Medicine
(V.P., D.V.), Medicine (J.G.L.), Chemical and Environmental Engineering (M. Marques dos Santos), and Soil, Water, and Environmental
Science (J.W.N., R.M.M.), University of Arizona, and the Arizona Respiratory Center and Bio5 Institute (J.G., V.P., S.E.M., J.G.L.,
F.D.M., D.V.) — all in Tucson; the Department of Occupational and Environmental Health, University of Iowa, Iowa City (N.M., P.S.T.);
Allergy and Asthma Consultants, Indianapolis (M.H.); and Dr. von Hauner Children’s Hospital, Ludwig Maximilians University Munich,
Munich, Germany (E.M.).
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