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Department of Biosciences, University of Helsinki, FI-00014 Helsinki, Finland; bSkin and Allergy Hospital, Helsinki University Central Hospital, FI-00029
Helsinki, Finland; cFinnish Institute of Occupational Health, FI-00250 Helsinki, Finland; dInstitute of Biotechnology, University of Helsinki, FI-00014 Helsinki,
Finland; eNational Institute for Health and Welfare, FI-00271 Helsinki, Finland; and fDepartment of Bacteriology and Immunology, Haartman Institute,
University of Helsinki, FI-00014 Helsinki, Finland
Contributed by Ilkka Hanski, April 4, 2012 (sent for review March 14, 2012)
Author contributions: I.H., L.v.H., M.J.M., E.V., T.U.K., and T.H. designed research; N.F., K.K.,
K.T., P.A., and L.P. performed research; N.F., K.K., and H.A. contributed new reagents/
analytic tools; T.L. contributed data on allergies; I.H., K.K., T.L., P.K., P.A., and L.P. analyzed
data; and I.H., L.v.H., and T.H. wrote the paper.
The authors declare no conict of interest.
Freely available online through the PNAS open access option.
Data deposition: The sequences reported in this paper have been deposited in the Sequence Read Archive at the European Bioinformatics Institute (accession no. ERP001059).
1
www.pnas.org/cgi/doi/10.1073/pnas.1205624109
Relative abundance
Bacterial class
Actinobacteria
Bacilli
Clostridia
Betaproteobacteria
Alphaproteobacteria
Gammaproteobacteria
No. of study subjects
Generic diversity
Percentage
Sign
No. of
genera
56.7
15.6
4.5
9.6
3.8
2.8
118
+
+
0.08
0.84
0.26
0.13
0.51
0.83
116
126
68
56
62
76
52
118
Sign
+
+
+
+
0.04
0.94
0.58
0.57
0.76
0.0003
116
those living in built areas and near water bodies. Repeating the
PCA for the relative abundances of the bacterial classes instead of
their generic diversity did not yield a signicant association with
PC1env, although the relative abundances of all proteobacterial
classes were positively correlated, and the relative abundances of
the other bacterial classes were negatively correlated, with PC1env,
consistent with the pattern found for generic diversity (PC2bac).
We used 15 vegetation and other land cover types to describe
the yards around the homes, with an average area of 0.17 ha. The
second principal component of these data was correlated with
PC2bac (P = 0.02), but because this factor was also correlated with
PC1env, it did not make an independent contribution in a multiple
regression model. PC2bac was not related to plant species richness
in the yard or to the type, age, or condition of the house.
Determination of Atopy. The study subjects were divided into
healthy individuals and atopic individuals based on IgE antibody
level in a screen with Phadiatop, a mixture of common inhalant
allergens. The distribution of IgE values was bimodal (SI Appendix,
Hanski et al.
Fig. S2), and in this this study we used a cutoff value of 2.5 kUA/L to
identify individuals with atopic sensitization. To verify that the
results were not sensitive to the exact cutoff point, we repeated the
association analyses involving atopy with a lower cutoff point of 1
kUA/L (SI Appendix, Fig. S2). The same study subjects had participated in a comprehensive study of allergy 7 y earlier, including
skin prick testing (SPT) against common inhalant allergens (14).
Evaluation of atopic sensitization by SPT in 2003 and an IgE screen
in 2010 yielded highly concordant results (2 = 63.2, P < 0.0001;
88% of individuals classied similarly), indicating constancy of
individuals atopic sensitization from early to late adolescence.
Environmental Biodiversity and Atopy. Atopic individuals resided
throughout the study area (SI Appendix, Fig. S3), with no spatial
autocorrelation in their occurrence in the study population
(Morans I, Z = 0.75, P = 0.45). Atopy was somewhat more
frequent in the small town of Joensuu than in the rest of the
study area (0.51 vs. 0.38), but the difference was not statistically
signicant (2 = 1.97, P = 0.16). However, atopy was signicantly explained by environmental biodiversity around the homes
of the study subjects. Atopy decreased with PC1env of the land
use types, and thus with the amount of forested and agricultural
land within 3 km of the home (Table 2). The types of vegetation
in the yard had no effect, but species richness of one group of
plantsuncommon native owering plantswas signicantly
negatively correlated with atopy (Table 2). This nding is illustrated in Fig. 2A, showing that the number of uncommon native
owering plant species was 25% higher in the yards of healthy
individuals compared with the yards of atopic individuals. In this
analysis, we used the total number of all plant species identied
in the yard as a covariate to account for differences in size and
type of yard. The effect of uncommon native owering plant
species was an exception, given that species richness in other
plant categories was unrelated to atopy (SI Appendix, Table S3).
Using the data collected in 2003 (14), we examined a number
of additional factors that might have affected atopy in the study
population (e.g., family member smoking, living on a farm, frequent contact with pets). None of these factors was signicantly
related to atopy (SI Appendix, Table S4).
Skin Microbiota and Atopy. Atopy was not related to PC2bac, which
we used to characterize the skin microbiota, but a strikingly
strong, more specic association was detected at the level of
bacterial class. Atopic individuals had highly signicantly (P =
0.0003) lower generic diversity of gammaproteobacteria on the
skin compared with healthy individuals (Fig. 2B and Tables 1 and
2). In this analysis, we used the total number of bacterial genera in
the sample to account for variation in sample size (i.e., number of
sequences), which is more effective here than using rarefaction
(SI Appendix). Note that the correlation was detected specically
with generic diversity, rather than with relative abundance of
gammaproteobacteria (Table 1). For the other bacterial classes,
there were no correlations that even approached signicance with
either generic diversity or relative abundance (Table 1). IgE tests
for specic common inhalant allergens (cat, dog, horse, birch,
timothy grass, and mugwort) were all negatively correlated with
the generic diversity of gammaproteobacteria, but none of these
correlations was much stronger than the others, with P values
ranging from 0.01 to 0.10 (SI Appendix, Table S5). These results
imply that the strong and highly signicant negative correlation
between the generic diversity of gammaproteobacteria and atopy
(Fig. 2B) is not due solely to any particular allergen.
We repeated the analysis of the correlation of atopy with
environmental biodiversity and the richness of gammaproteobacteria using two other criteria for atopy: a different cutoff
point for IgE values (SI Appendix, Fig. S2) and the SPT done in
2003. The results remained essentially the same (SI Appendix,
Table S6).
PNAS | May 22, 2012 | vol. 109 | no. 21 | 8335
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Regression model
Deviance
Difference
Coefcient
158.0
117.6
107.4
100.9
40.4
10.1
6.5
<0.0001
0.0014
0.011
0.58
0.52
0.10
0.31
0.023
0.0059
0.0016
0.015
0.20
38/94
The three explanatory variables are the rst principal component of land use types (PC1env; SI Appendix,
Table S1), the number of uncommon native owering plant species in the yard (residual, accounting for the
effects of eld assistants and the total number of vascular plant species in the yard; Fig. 2A), and the generic
diversity of gammaproteobacteria (residual, accounting for the effect of the total number of bacterial genera; Fig. 2B).
Gammaproteobacteria and Immune Function. The foregoing observational results (Fig. 2B and Tables 1 and 2) imply a strong
allergy-protective effect for gammaproteobacteria. This conclusion is supported by in vitro measurements of baseline levels of
IL-10 mRNA in PBMCs in a large subset of the study subjects
(n = 69). We analyzed correlations between IL-10 expression
and the relative abundance and generic diversity of the different
bacterial classes in the skin microbiota separately for healthy
individuals and atopic individuals. We found one signicant
correlation, between the relative abundance of gammaproteobacteria and IL-10 expression in healthy individuals (P = 0.015)
(SI Appendix, Table S7). Examining this association at the generic level revealed one highly signicant correlation, between
the relative abundance of Acinetobacter and IL-10 expression
(P = 0.0004) (SI Appendix, Table S8), which was reversed in
atopic individuals (Fig. 3). At present, we do not know which
cells are mainly responsible for IL-10 secretion in this material.
In addition to allergen-specic T regulatory cells (15), T effector
cells, B cells, and innate cells (monocytes/macrophages) (16)
secrete substantial amounts of IL-10 and thus potentially inuence the development of immunologic tolerance.
Discussion
Environmental biodiversity, the human commensal microbiota,
and the human immune system are complex systems with numerous
Fig. 2. Relationships among environmental biodiversity, skin microbiota, and atopy in the study subjects. (A) The number of uncommon native owering
plant species plotted against the total number of plant species in the yard of atopic individuals (solid symbols) and healthy individuals (open symbols). The
number of plant species on the vertical axis is the residual accounting for variation in the results of ve pairs of eld assistants. The effect of atopy is signicant (t = 3.14, P = 0.0022, n = 94; without correcting for the effect of eld assistants, t = 2.83, P = 0.0058). (B) The number of genera of gammaproteobacteria plotted against the total number of bacterial genera in the skin microbiota of atopic individuals (solid symbols) and healthy individuals (open
symbols). The effect of atopy is highly signicant (t = 3.72, P = 0.0003, n = 112).
8336 | www.pnas.org/cgi/doi/10.1073/pnas.1205624109
Hanski et al.
Fig. 4. Summary graph of the associations among environmental biodiversity, skin microbiota, and atopy. The solid arrows refer to the results in
Figs. 13 and Table 2. The dashed-line arrow indicates a less signicant effect
of PC1env on the generic diversity of gammaproteobacteria (t = 1.91, P = 0.059,
n = 95, with total number of bacterial genera as a covariate as in Fig. 2B).
Hanski et al.
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Fig. 3. Cytokine IL-10 expression against the relative abundance of Acinetobacter in the skin community of healthy (open symbols) and atopic individuals (lled dots). The interaction term is highly signicant (P = 0.0009 in
a linear regression model, with adjusted R2 = 0.23) (SI Appendix, Table S8).
study of allergy in 2003 (14), and their families continued to live, with a few
exceptions, in the same dwellings in 20102011, when data and samples for
the present study were collected. The subjects homes included apartments,
row homes, and individual houses in the town of Joensuu (73,000 inhabitants) and villages of varying populations, as well as isolated houses in the
sparsely populated countryside (SI Appendix, Fig. S3).
Allergy Tests. Blood samples (n = 116) were collected in September 2010 and
screened with Phadiatop (a mixture of common inhalant allergens) and
ImmunoCAP (Phadia). Samples with an IgE antibody level 0.35 kUA/L were
also analyzed for allergen-specic IgE antibodies. In the previous study of
the same subjects in 2003, SPT was performed against a standard set of eight
inhalant allergens (ALK-Abello) with negative (solvent) and positive controls
(histamine dihydrochloride 10 mg/mL). Individuals positive (i.e., wheal diameter 3 mm) for at least one of the eight inhalant allergens tested were
classied as atopic. Background and clinical data were obtained using
questionnaires (14). The characteristics of the study subjects with respect to
atopy as dened by SPT are presented in SI Appendix, Table S4.
Real-Time Quantitative PCR Analysis. Blood samples (n = 69) were collected in
September 2011. PBMCs were separated from whole blood in BD Vacutainer
CPT cell preparation tubes with sodium heparin, frozen, and shipped to the
analysis site. This approach provides unique opportunities to stimulate cells
in vitro by different compounds, as well as to analyze cytokine and surface
molecule expression using state-of-the-art biomedical methods. The thawed
PBMCs were cultured in 24-well plates at 1 106/mL in complete RPMI-1640
medium with 10% heat-inactivated FBS at 37 C and 5% CO2. After 24 h of
culture, total RNA was extracted from the PBMCs using Trisure (Bioline)
according to the manufacturers instructions. The RNA was reverse-transcribed into cDNA, and the mRNA expression level of IL-10 was measured by
real-time quantitative PCR using the Applied Biosystems 7500 Fast Real-Time
PCR System. PCR amplication of the endogenous 18S rRNA was performed
for each sample to control sample loading and allow normalization between
the samples. The results were expressed as relative units, calculated by the
comparative CT method according to the manufacturers instructions.
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