Aberrant IgA responses to the gut microbiota
during infancy precede asthma and allergy
development
Majda Dzidic, Thomas Abrahamsson, Alejandro Artacho, Bengt Björksten, Maria Carmen Collado, Alex Mira and Maria Jenmalm
Journal Article
N.B.: When citing this work, cite the original article. Original Publication:
Majda Dzidic, Thomas Abrahamsson, Alejandro Artacho, Bengt Björksten, Maria Carmen Collado, Alex Mira and Maria Jenmalm, Aberrant IgA responses to the gut microbiota during infancy precede asthma and allergy development, Journal of Allergy and Clinical Immunology, 2017. 139(3), pp.1017-+.
http://dx.doi.org/10.1016/j.jaci.2016.06.047 Copyright: Elsevier
http://www.elsevier.com/
Postprint available at: Linköping University Electronic Press
Aberrant IgA responses to the gut microbiota during infancy precedes asthma and allergy development
Majda Dzidic, MSc, Thomas R. Abrahamsson, MD, PhD, Alejandro Artacho, BSc, Bengt Björkstén, MD, PhD, Maria Carmen Collado, PhD, Alex Mira, PhD, Maria C. Jenmalm, PhD
PII: S0091-6749(16)30786-2
DOI: 10.1016/j.jaci.2016.06.047
Reference: YMAI 12276
To appear in: Journal of Allergy and Clinical Immunology
Received Date: 1 March 2016 Revised Date: 11 May 2016 Accepted Date: 8 June 2016
Please cite this article as: Dzidic M, Abrahamsson TR, Artacho A, Björkstén B, Collado MC, Mira A, Jenmalm MC, Aberrant IgA responses to the gut microbiota during infancy precedes asthma and allergy development, Journal of Allergy and Clinical Immunology (2016), doi: 10.1016/j.jaci.2016.06.047. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Aberrant IgA responses to the gut microbiota during
1
infancy precedes asthma and allergy development
2
Authors: Majda Dzidic, MSc1,3,5, Thomas R. Abrahamsson, MD,PhD2, Alejandro
3
Artacho, BSc3, Bengt Björkstén, MD, PhD4, Maria Carmen Collado, PhD5, Alex
4
Mira, PhD3†*, Maria C. Jenmalm, PhD1†*
5 6
Affiliations:
7
1
Department of Clinical and Experimental Medicine, Unit of Autoimmunity and
8
Immune Regulation, Linköping University, Linköping, Sweden
9
2
Department of Clinical and Experimental Medicine, Division of Paediatrics,
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Linköping University, Linköping, Sweden
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3
Department of Health and Genomics, FISABIO Foundation, Center for Advanced
12
Research in Public Health, Valencia, Spain
13
4
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
14
5
Institute of Agrochemistry and Food Technology, Spanish National Research
15
Council (IATA-CSIC), Department of Biotechnology, Unit of Lactic Acid Bacteria
16
and Probiotics, Valencia, Spain
17 18
† These authors share senior authorship based on equal contribution.
19
* To whom correspondence should be addressed:
20
Maria Jenmalm
21
Address: Linköping University, Department of Clinical and Experimental Medicine, 22
AIR/Clinical Immunology, 581 85 Linköping, Sweden. 23
Email address: maria.jenmalm@liu.se 24 Phone number: +46 101034101 25 Fax +46-13-13 22 57 26 Alex Mira 27
Address: Avenida de Cataluna 21, 46020 Valencia, Spain. 28
Email address: mira_ale@gva.es 29
Phone number: +34 961925925 30
31 32
Funding: Supported by the Swedish Research Council (K2011-56X-21854-01-06);
33
the Swedish Heart-Lung Foundation (20140321); the Ekhaga Foundation (210-53);
34
the Medical Research Council of Southeast Sweden; the Olle Engqvist Foundation;
35
the Cancer and Allergy Foundation; the University Hospital of Linköping, Sweden;
36
and by the Grant 2012-40007 from Spanish MINECO to Alex Mira.
37 38 39 40
Conflicts of interest: Maria Jenmalm and Thomas Abrahamsson have received
41
honoraria for lectures and funding for a clinical trial from Biogaia AB, Sweden. The
42
rest of the authors declare that they have no relevant conflicts of interest.
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Abstract
44 45Background: While a reduced gut microbiota diversity and low mucosal total IgA
46
levels in infancy have been associated with allergy development, IgA responses to the
47
gut microbiota have not yet been studied.
48
Objective: We sought to determine the proportions of IgA coating together with the
49
characterization of the dominant bacteria, bound to IgA or not, in infant stool samples
50
in relation to allergy development.
51
Methods: A combination of flow cytometry cell sorting and deep sequencing of the
52
16S rDNA gene was used to characterize the bacterial recognition patterns by IgA in
53
stool samples collected at 1 and 12 month of age from children staying healthy or
54
developing allergic symptoms up to seven years of age.
55
Results: The children developing allergic manifestations, particularly asthma, during
56
childhood had a lower proportion of IgA bound to fecal bacteria at 12 months of age
57
compared with healthy children. These alterations cannot be attributed to differences
58
in IgA levels or bacterial load between the two groups. Moreover, the bacterial targets
59
of early IgA responses (including the coating of Bacteroides genus) as well as the IgA
60
recognition patterns differed between healthy children and children developing
61
allergic manifestations. Altered IgA recognition patterns in children developing
62
allergy were observed also already at 1 month of age, when the IgA antibodies are
63
predominantly maternally derived in breast fed children.
64
Conclusion: An aberrant IgA responsiveness to the gut microbiota during infancy
65
precedes asthma and allergy development, possibly indicating an impaired mucosal
66
barrier function in allergic children.
67
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Key message: Aberrant and reduced IgA responses to the gut microbiota during
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infancy precede development of asthma and allergic disease during the first seven
70
years of life.
71
72
Capsule summary: Early characterization of IgA coating patterns may represent a
73
novel way to identify infants with increased risk to develop asthma and allergic
74
disease. Moreover, interventions enhancing infant mucosal barrier function may
75
represent efficacious preventive strategies required to combat the asthma and allergy
76
epidemic.
77 78
Key words: Allergic disease; asthma; SIgA; IgA index; IgA recognition patterns;
79
microbiome composition; gut microbiota, childhood.
80 81 Abbreviations used: 82 A: Allergic 83
ARC: Allergic rhinoconjunctivitis
84
H: Healthy
85
IgA: Immunoglobulin A
86
IgA+: IgA coated
87
IgA-: non IgA bound
88
PCA: Principal Component Analysis
89
RDP: Ribosomal Database Project
90
SIgA: Secretory immunoglobulin A
91
TLR: Toll Like Receptor
92
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Introduction
94Allergic diseases have become a major public health problem in affluent societies.(1)
95
Reduced microbial exposure, both pre- and postnatally, has been proposed to underlie
96
the increase in allergy development.(2-5) The gut microbiota, hosting a complex
97
bacterial community, is quantitatively the most important source of microbial
98
stimulation and may provide a primary signal for appropriate immune
99
development.(4) The gut microbiota differs in composition and diversity during the
100
first months of life in children who later do or do not develop allergic disease,(2,6-17)
101
although no specific microbes with consistently harmful or allergy protective roles
102
have yet been identified. Also, we observed that the differences in the gut microbiota
103
diversity during infancy between healthy children and children developing allergies
104
were mainly related to asthma and not allergic rhinoconjunctivitis development.(16)
105
Early establishment of a diverse gut microbiota, with repeated exposure to new
106
bacterial antigens, may be more important than the distribution of specific microbial
107
species in shaping a normal immune mucosal and systemic maturation.(4)
108 109
A reduced mucosal barrier function may increase the risk for allergy development(1)
110
and immunoglobulin A (IgA) is the primary mediator of humoral mucosal
111
immunity.(18) Immunoglobulin A is the most abundantly produced antibody in
112
humans, with the highest amount of secretion in the intestinal tract.(18,19) Secretory
113
IgA (SIgA) has a crucial role in the gut through its binding to bacterial antigens, thus
114
preventing their direct interaction with the host via immune exclusion and
115
maintaining the mucosal homeostasis.(18,20) SIgA may also limit overgrowth of
116
select species, thus stimulating diversity.(18,21) Therefore, this antibody represents a
117
key host mechanism in regulation of the commensal community, and innate receptor
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signaling in T-cells seems to decide the specificity of IgA to constrain the
119
composition of the intestinal bacteria, ensuring a benign symbiotic relationship.(19)
120
However, in contrast to IgG and IgM levels, the generation of this anti-inflammatory
121
antibody is limited during early infancy and delayed development of mucosal IgA
122
production, for instance in the absence of breastfeeding, may lead to infectious
123
disease in young infants.(22,23) Studies and clinical reports suggest that SIgA that
124
origins from the mothers’ breast milk is important for immune regulation and
125
protection against bacterial, viral and parasitic infections in suckling infants.(23-27)
126
Whereas total levels of SIgA in saliva and fecal samples have been investigated in
127
children developing allergy before, little is known about the identities of the bacterial
128
taxa targeted by IgA in the infant gut and what role mucosal immune responses to the
129
gut microbiota plays in childhood allergy development. However, earlier studies have
130
shown that low levels of salivary and intestinal SIgA are associated with an increased
131
risk for allergic manifestations during early life.(28-30) Recent advances in flow
132
cytometry(31) and next generation sequencing(32) now allow studying the complex
133
interactions between human antibodies and microbiota. In this study, we have used
134
flow cytometry-based cell sorting and barcoded 16S rDNA 454-pyrosequencing to
135
characterize the dominant gut bacteria, coated or non-coated with IgA, and
136
determined total secretory IgA levels and bacterial load in stool samples collected
137
during the first year of life in infants who either developed allergic manifestations or
138
stayed healthy up to 7 years of age.
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Methods
140For detailed methods, experimental protocols and statistical analyses, see the Methods
141
section in this article's Online Repository at www.jacionline.org.
142
143
Study design
144
The infants included in this study were part of a larger randomized double-blind trial
145
in South-Eastern Sweden between 2001 and 2003, evaluating the potential allergy
146
prevention effect of probiotic Lactobacillus reuteri ATCC 55730, until 2(33) and 7
147
years of age.(34) The recruited children had a family history of allergic disease (1 or
148
more family members with eczema, asthma, gastrointestinal allergy, allergic urticaria
149
or allergic rhinoconjunctivitis), and more detailed inclusion and exclusion criteria are
150
explained in the study of Abrahamsson et al.(33) Among the 188 infants completing
151
the original study, infant stool samples collected at 1 and 12 months of life in 20
152
children developing allergy (Table EI in this article's Online Repository at
153
www.jacionline.org) and 28 children staying healthy up to 7 years of age (Table EII),
154
were randomly selected for this study (Fig. 1). Ten of the allergic children developed
155
asthma. Other allergic diseases included eczema (n=9 at 7 years of age; n=17 at 2
156
years of age; no infants developed eczema before 1 month of age), allergic
157
rhinoconjunctivitis (n=10) and allergic urticaria (n=1), with symptoms defined as
158
described in detail previously.(33,34) The samples were immediately frozen at -20°C
159
following collection and later stored at -70°C until use.
160 161
There were no differences regarding potential confounders, such as sex, mode of
162
delivery, birth order, maternal atopy, breastfeeding, antibiotics, and probiotic
163
supplementation, between the infants who did or did not develop allergic
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manifestations (Table I). All included infants were exclusively breast-fed for at least 1
165
month, and no infant received antibiotics before 1 month of age.(12) The Regional
166
Ethics Committee for Human Research at Linköping University approved the study.
167
Informed consent was obtained from both parents before inclusion. The study is
168
registered at ClinicalTrials.gov (ID NCT01285830).
169 170
Sample labeling and flow cytometry protocol
171
Stool samples were suspended in sterile saline solution (autoclaved H2O; NaCl
172
Sodium Chloride 99.5% PA-ACS-ISO, Panreac; Barcelona, Spain; Ref. 131689.1211)
173
with 5% bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA; Ref.
A7030-174
100gr) in order to prevent non-specific antibody binding. The samples were stained
175
with goat anti-mouse IgA labeled with FITC, used as an isotype control
176
corresponding to unspecific binding, (Invitrogen, Frederick, MD, USA; Ref. M31001)
177
or with goat anti-human IgA labeled with FITC (Invitrogen; Ref. H14001), according
178
to the manufacturer instructions (Fig. E1 in this article’s Online Repository at
179
www.jacionline.org). The sorting of the bacterial cells according to whether they were
180
IgA coated (IgA+) or IgA non-coated (IgA-) was performed by MoFloTM XDP Cell
181
Sorter (Beckman Coulter, Inc; Brea, CA, USA), following Simon-Soro et al.
182 2015.(32) 183 184 DNA-extraction 185
DNA from sorted fecal bacteria, IgA+ and IgA-, was isolated using the MasterPureTM
186
complete DNA and RNA Purification Kit (Epicentre Biotechnologies, Madison, WI,
187
USA), following the manufacturer’s instructions with a previous glass bead beating
188
(0.17 mm diameter) and an additional enzymatic lysis step with lysozyme (20mg/ml,
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37°C, 30 min; Thermomixer comfort, Eppendorf, Hamburg, Germany).
190
191
16S rDNA gene amplification and sequencing
192
DNA from 192 samples in total was used for PCR amplification and pyrosequencing
193
in order to describe bacterial composition of the sorted populations. A region of
194
approximately 650-bp of the 16S rDNA gene was amplified using universal bacterial
195
degenerate primers(35), which encompass the hypervariable regions V3-V5 of the
196
gene. A secondary amplification was performed by using the purified PCR product as
197
a template.(36)
198
199
Sequence processing and taxonomic classification
200
The resulting 16S rDNA read ends were trimmed in 10 bp sliding windows, with
201
average value ≥20, using the Galaxy tool(37) and only reads longer than 250 bp were
202
considered. The sequences were assigned to each sample by the 8 bp barcode through
203
the Ribosomal Database Project (RDP) pipeline(38) version 11.3, and chimeric
204
sequences were filtered out using UCHIME.(39)
205 206
Taxonomic assignment was performed by the RDP-classifier(38) where the reads
207
were assigned a phylum, class, family and genus and phylogenetic ranks were
208
allocated when scores exceeded 0.8 confidence threshold. Shannon indices, based on
209
randomly selected 700 reads per sample, was utilized to estimate the samples’
210
diversity on gene and phylum level.
211 212
For analyzing IgA coating patterns, the threshold used for including the genera was
213
≥1% relative abundance in either the IgA+ or IgA- fractions. The abundance
214
proportions of a given genera was used to calculate the ratio between IgA+ and IgA-
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fractions, giving the IgA index.(40) Thus, this score was based on proportional
216
representation, for every given genus, within the IgA+ (positive IgA index values)
217
and IgA- fractions (negative IgA index values), reflecting the degree of mucosal
218
immune responsiveness to the microbiota. LDA Effect Size (LEfSe)(41) was then
219
used for high-dimensional biomarker discovery comparing the IgA-indices between
220
healthy infants and infants developing allergic manifestations. Furthermore, Principal
221
Component Analyses (PCA) was performed by R software ade4 package.(42)
222 223
Bacterial load analysis with qPCR
224
qPCR amplifications were performed in order to measure the bacterial load (number
225
of bacterial cells normalized by the number of human cells) using primers targeting
226
the single-copy housekeeping bacterial gene FusAand the human β-actin gene (Table
227
EIII).
228 229
Determination of secretory IgA concentrations in stool samples
230
A commercially available ELISA kit was used for the determination of total secretory
231
IgA concentrations in feces samples (ImmuChrom ELISA kit, ImmuChrom GmbH,
232
Heppenheim, Germany) following the manufacturer’s instructions.
233 234
Statistics
235
Statistical analyses were performed in R version 3.2.2 and GraphPad Prism 6
236
(GraphPad Software, San Diego, CA, USA, Version 6.1f), where p<0.05 was
237
considered significant. For a more comprehensive description of the statistical
238
methods, please see this article’s Online Repository at www.jacionline.org.
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Results
240 241Proportion of fecal bacteria bound to IgA in relation to allergy development 242
Infants developing allergic symptoms during the first 7 years of life had significantly
243
lower proportions of IgA-coated fecal bacteria at 12 months of age than healthy
244
children (Fig. 2A), while similar proportions were observed at 1 month of age. A low
245
proportion of IgA-coated fecal bacteria at 12 months of age also preceded
246
development of asthma (Fig. 2B), but not allergic rhinoconjunctivitis (Fig. E2 in this
247
article’s Online Repository at www.jacionline.org). Moreover, independently of
248
allergy development, an overall decreasing proportion of fecal bacteria bound to IgA
249
from 1 to 12 months of age was observed, likely reflecting a change from
250
predominantly maternally breast milk derived to child derived IgA antibodies.(22,43)
251
252
The influence of possible confounding factors was also evaluated. However,
253
supplementation of the probiotic bacterium L. reuteri, delivery mode, antibiotic
254
treatments and partial breastfeeding at 12 months of age did not affect the proportion
255
of IgA-coated fecal bacteria (Fig. E3).
256 257
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Bacterial load, but not total SIgA levels, differ in healthy children and children 258
developing allergic manifestations 259
In order to better understand detected differences in IgA proportions between healthy
260
children and children developing allergic manifestations, bacterial load and total SIgA
261
levels in stool samples were measured. The bacterial load was higher at 12 months of
262
age in children staying healthy than in those developing allergic manifestations (Fig.
263
3A), but not significantly so for asthma (p=0.11) and ARC (p=0.61). A similar
264
bacterial load was observed at 1 month of age in children staying healthy and
265
developing allergy (Fig. 3A).
266 267
Total fecal SIgA levels were similar in healthy children and children developing
268
allergic manifestations (Fig. 3B), asthma (p=0.38 and p=0.71, for 1 and 12 months,
269
respectively) and allergic rhinoconjunctivitis (p=0.77 and p=0.78, for 1 and 12
270
months, respectively). The fecal SIgA levels decreased significantly from 1 to 12
271
months of age in both groups (Fig. 3B).
272 273
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Bacterial targets of IgA responses in children developing allergic manifestations 274
and children staying healthy up to 7 years of age 275
Bacterial 16S rDNA gene sequencing of IgA+ and IgA- fractions was performed in
276
order to assess early IgA responses in children staying healthy and children
277
developing allergic manifestations during the first 7 years of age. After quality
278
filtering and removal of chimeric sequences, 190 samples with 633,378 high-quality
279
sequence reads remained, with an average of 3,316 reads per sample and a mean
280
length of 515 bp.
281
While the analysis of the relative abundance of dominant bacterial families was
282
generally similar between children developing allergy and staying healthy (Fig. E4 in
283
this article’s Online Repository at www.jacionline.org), clear differences were
284
observed upon analyzing the bacterial targets of IgA responses, represented as IgA
285
index. IgA responses to the gut microbiota were demonstrated to differ between
286
healthy children and children developing allergic manifestations (Fig. 4A, B) and
287
asthmatic symptoms, particularly at 1 month but also 12 months of age (Fig. 4C, D).
288
At 1 month of age the genus Faecalibacterium was mainly IgA free (IgA-) in children
289
developing allergic manifestations (including asthma but not allergic
290
rhinoconjunctivitis, Fig. E5A). Moreover, the genera Parabacteroides and
291
Anaerococcus were primarily not targeted by IgA in children developing allergic
292
manifestations, when compared with healthy children.
293
At 12 months of age, the IgA responses of children developing allergic manifestations
294
were mainly not targeting the Bacteroides genus (Fig. 4B), with similar findings
295
observed for children developing allergic rhinoconjunctivitis (Fig. E5B). Regarding
296
children developing asthmatic symptoms, Escherichia/Shigella was predominantly
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IgA free (Fig. 4D), while Lachnospiraceae incertae sedis, a Firmicutes phylum
298
member, was predominantly IgA bound in children developing allergic symptoms,
299
when compared with healthy children (Fig. 4B). Furthermore, the genera Roseburia
300
and Erysipelotrichaceae incertae sedis were generally IgA-targeted in healthy
301
children, but not in children showing allergic manifestations during the first 7 years of
302
life. In contrast, decreased IgA responses to the Veillonella genus was observed in
303
healthy children.
304
Possible differences in bacterial diversity in children developing allergy and staying
305
healthy were also of interest. Thus, Shannon indices for IgA+ and IgA- fractions were
306
calculated. No differences, neither at genus (Fig. E6) or at phylum level (Fig. E7A-B)
307
were found in relation to allergy development, however, except that children
308
developing asthma had increased diversity at 12 months among IgA coated
309
Bacteroidetes and Proteobacteria (Fig. E7C) but not in the IgA-free fraction (Fig.
310
E7D).
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IgA recognition patterns of gut microbiota differ between healthy and allergic 312
children 313
A principal components analysis (PCA), based on the calculated IgA indices, was
314
used in order to evaluate the differences in IgA responses to the gut microbiota
315
between healthy children and children developing allergic manifestations, including
316
asthma. Interestingly, the IgA recognition patterns differed already at 1 month of age
317
when comparing healthy children and children developing allergic (Fig. 5A) and
318
asthmatic symptoms (Fig. 5C) but not ARC (Fig. E8A in this article’s Online
319
Repository at www.jacionline.org). Clear separation was observed at 12 months of
320
age for healthy children and children developing allergic disease (Fig. 5B), asthma
321
(Fig. 5D) and ARC (Fig. E8B).
322
No effect of potential confounding factors (probiotic supplementation, the delivery
323
mode, antibiotic treatment and partial breastfeeding at 12 months) on IgA recognition
324
was observed (Fig. E9).
325 326
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Discussion
327The data presented in the current study demonstrate that the first year of life
328
represents an early-life critical period in which aberrant gut microbiota IgA responses
329
are linked to the risk of developing asthma and allergic disease. SIgA functions as a
330
first line of defense by interfering with the microbiota and thus protect the intestinal
331
tissue from invasion and destruction by pathogenic and commensal bacteria.(18,44)
332
Thus, intact production and function of IgA is a key mechanism to preserve intestinal
333
health by directly influencing the properties of the microbiota and enhancing mucosal
334
barrier function.(18) Furthermore, SIgA may also limit overgrowth of selected
335
species, thus enabling an increased microbiota diversity.(18,21) This can be
336
particularly crucial during early childhood, when the microbiota plays a central role in
337
immune modulation and where microbial recognition by maternal and infant
338
antibodies must be appropriately orchestrated for an optimal maturation of the
339
immune system.(45-47) In line with this, low mucosal total IgA levels(28-30) a
340
reduced gut microbiota diversity in infancy(8-16) and decreased seroreactivity to gut
341
microbiota antigens(48) have been associated with allergy development. However,
342
intestinal IgA responses to the infant gut microbiota have not previously been studied
343
in relation to allergy development. To investigate this, we have used a combination of
344
flow cytometry and high-throughput deep sequencing to characterize the patterns of
345
bacterial recognition by IgA in stool samples collected at 1 and 12 month of age from
346
children staying healthy or developing allergic symptoms up to seven years of age.
347
348
Interestingly, development of allergic disease, particularly asthma, during childhood
349
was associated with a reduced proportion of IgA bound to fecal bacteria at 12 months
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of age. To better understand these differences, we sought to investigate if the bacterial
351
load and total fecal IgA levels might be of any influence. The results showed that the
352
lower proportions of IgA coated fecal bacteria among children developing allergic
353
manifestations were independent of total fecal SIgA levels that were relatively similar
354
to healthy children, especially at 12 months of age. Moreover, the decreased
355
proportions of IgA coated bacteria in allergic children are probably not due to lower
356
IgA antibodies-to-bacteria ratios because bacterial densities were actually lower in
357
children developing allergies. Thus, in addition to the lower bacterial diversity
358
detected by other studies,(8-16) allergic children seem also to be exposed to lower
359
microbial densities in the gut. These two factors could lead to decreased stimulation
360
of the immune system via TLR:s,(19) affecting the production and microbial
361
recognition patterns of IgA, thus leading to lower proportion of IgA coating in
362
allergic than healthy children. It would be interesting to further investigate the role of
363
factors influencing IgA production, such as vitamin A-derived retinoic acid, TGF-b,
364
IL-10, BAFF and APRIL(49), in the aberrant IgA responses in children developing
365
allergy in future studies. As we previously found that a low gut microbiota diversity
366
in infancy was mainly related with asthma, but not allergic rhinoconjunctivitis,
367
development at school age(16), we here aimed to determine the importance of IgA
368
responses to the gut microbiota particularly for asthma development. Speculatively,
369
the association with asthma could be due to the fact that viral lower respiratory tract
370
infections have been linked to asthma development among atopic children.(1,50,51)
371
Thus, low IgA responses to the microbiota may result in a reduced mucosal barrier
372
function. This may cause an increased susceptibility to airway viral infections, leading
373
to amplification of Th2 responses and subsequent asthma development.(1,50,51)
374
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Previous studies in adults have determined that IgA might be more reactive against
376
disease driving bacteria,(44,52-54) in line with the theory that the immune system can
377
distinguish between pathogens and commensals through sensing pathogen-associated
378
behaviors, including adherence to the intestinal epithelium and tissue invasion.(53,54)
379
Also, SIgA may enable an increased microbiota diversity by limiting overgrowth of
380
selected species.(18,21) In the current study, we observed that the IgA recognition
381
patterns differed between healthy children and children developing allergic
382
symptoms, including asthmatic disease and allergic rhinoconjunctivitis, with clearly
383
divergent IgA index patterns already at 1 month of age. As the IgA antibodies at 1
384
month of age in exclusively breast fed infants are predominantly maternally
385
derived,(22,43) the divergent responses observed at this time point suggest that the
386
immunological interactions between mother and offspring influence allergy
387
development, in line with previous studies.(45-47) For example, breast milk derived
388
SIgA had a large impact on microbial colonization in neonatal mice and was crucial
389
for healthy intestinal epithelial barrier function and immune homeostasis in the
390
offspring.(25)
391
392
Interestingly, the gut commensals Faecalibacterium and Bacteroides were mainly
393
IgA free at 1 and 12 months of age in children showing allergic manifestations but
394
were predominantly IgA coated in healthy children, especially at 12 months. These
395
two genera are important human gut symbionts, involved in production of butyrate, an
396
end product of colonic fermentation that is important in maintaining a healthy
397
gut.(55-57) Furthermore, decreased diversity of the Bacteroidetes phylum in infant
398
stool samples have been linked to delivery by Cesarean section(4,58) and allergy
399
development.(4,12) Other commensals that seem to be ignored by the IgA recognition
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in children developing allergic symptoms were Parabacteroides, at 1 month of age,
401
and Roseburia, at 12 months of age. The Parabacteroides species distasonis have
402
been shown to reduce inflammatory responses in murine models with chronic
403
colitis,(59) while Roseburia is another well-known butyrate-producer(60), which was
404
reduced in patients suffering from ulcerative colitis.(61) In all, decreased coating of
405
these commensals might reflect a lower stimulation of the mucosal immune system in
406
the infants developing allergic diseases.
407 408
The Erysipelotrichaceae family is considered to be highly immunogenic and seems
409
important in inflammation related disorders of the gastrointestinal tract as they are
410
enriched in colorectal cancer.(62,63) Palm and colleagues found that a member of
411
Erysipelotrichaceae was highly coated by IgA in specific pathogen free mice, relative
412
to other members of the gut microbiota, proposing its role as a colitogenic
413
bacteria.(54) Furthermore, deregulation of T-cells in mice affecting the selection of
414
IgA plasma cells caused gut dysbiosis, including increased abundance of
415
Erysipelotrichaceae that are known to induce immune hyperactivation.(64)
416
Escherichia and Shigella genera are Proteobacteria proposed to express highly
417
proinflammatory hexa-acylated endotoxin production and were enriched in adult
418
asthmatic patients triggering airway inflammation.(65) Moreover, high abundance of
419
fecal Escherichia coli was associated with development of IgE-associated eczema
420
within the first year of life.(66) As allergy and asthma development were associated
421
with reduced IgA responses to the Erysipelotrichaceae and Escherichia/Shigella
422
genus respectively, at 12 months of age, this may suggest an impaired mucosal
423
immune exclusion of this genus in children developing allergic disease, possibly
424
leading to proinflammatory responses enhancing disease susceptibility.
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In contrast, allergy development was associated with increased IgA responses at 12
426
months of age to Lachnospiraceae, a Gram positive barrier associated microbes that
427
are colonizing the inner mucus layer, staying in close contact with host
428
mucosa.(67,68) Furthermore, excessive growth of species belonging to
429
Lachnospiracea in mice with impaired IgA responses reduces Firmicutes
430
diversity.(21) The increased IgA coating of these bacteria in children developing
431
allergies might thus be an indication of an altered mucosal barrier function.
432 433
Factors that might influence the development of the intestinal microbiota and the
434
mucosal immune system include the mode of delivery, exposure to antibiotics, partial
435
breastfeeding at 12 months of age and probiotic supplementation.(14,23,58,69,70)
436
These confounding factors seem not to have influence in our study population, since
437
the discovered differences are driven by health status. However, larger studies are
438
required to further investigate and confirm the role of these factors. Also, it needs to
439
be determined whether our findings can be replicated in cohorts of other geographic
440
origins and with different family history of allergic disease.
441
442
In conclusion, our work suggests that studies of IgA responses to gut microbiota
443
during infancy could be used to determine the normal development of mucosal
444
immunity and establishment of a healthy symbiosis with gut microbes, and how
445
maternal immunity affects these processes. Early characterization of IgA coating
446
patterns may represent a novel way to identify infants with increased risk to develop
447
asthma and allergic disease, although this needs to be confirmed in larger cohorts.
448
Furthermore, interventions enhancing infant mucosal barrier function may represent
449
efficacious preventive strategies required to combat the asthma and allergy epidemic.
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Acknowledgments: We thank Mrs Anne-Marie Fornander for excellent technical
451
assistance and Sandra Garcia Esteban for her great assistance in the laboratory work.
452
Funding: Supported by the Swedish Research Council (K2011-56X-21854-01-06);
453
the Swedish Heart-Lung Foundation (20140321); the Ekhaga Foundation (210-53);
454
the Medical Research Council of Southeast Sweden; the Olle Engqvist Foundation;
455
the Cancer and Allergy Foundation; the University Hospital of Linköping, Sweden;
456
and by the Grant 2012-40007 from Spanish MINECO to Alex Mira.
457 458
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Fig. 1. Schematic workflow of performed experiments. The proportion of the gut microbiota bound
459
to IgA (IgA+) or not (IgA-), in infants, was analyzed by flow cytometry-based sorting of fecal samples 460
prior to 16S rDNA 454-pyrosequencing. In addition, total secretory IgA levels were estimated by 461
ELISA tests, and the bacterial density measured using universal primers targeting the single-copy 462
bacterial gene FusA. 463
Fig. 2. Proportions of IgA-coated fecal bacteria in early infancy. A) The proportion of fecal bacteria
464
bound to IgA at 1 month and 12 months of age in children staying healthy (n=28, circles) or 465
developing allergic symptoms (n=20, triangles) during the first 7 years of life. B) The proportion of 466
fecal bacteria bound to IgA at 1 month and 12 months of age in children staying healthy (n=28, circles) 467
or developing asthma (n=10, triangles) during the first 7 years of life. Median and interquartile ranges 468
are indicated. *p < 0.05; **p < 0.01 (Mann–Whitney U-test). 469
Fig. 3. Bacterial load and total fecal secretory IgA levels in healthy infants and infants developing
470
allergic manifestations. A) The quantification of bacterial numbers was obtained by qPCR- detection
471
with universal primers targeting the gene FusA (present in single-copy in bacterial cells) and 472
normalized by the number of human cells, determined by qPCR- detection with primers for the human 473
β-actin gene. nHealthy=28; nAllergic=20. B) Total secretory IgA levels in stool samples were measured
474
using ELISA immunoassay. 1 month of age: nHealthy=25; nAllergic=19; 12 months of age: nHealthy=27;
475
nAllergic=19. Means with standard errors are indicated. * p<0.05; ** p<0.01; *** p<0.001 (Mann
476
Whitney U-test and Wilcoxon matched pairs test for unpaired and paired comparisons, respectively). 477
Fig. 4. IgA responses to the gut microbiota, at 1 and 12 months of age. Plots are depicting IgA
478
responses (defined by IgA index, reflecting the ratioin IgA+ and IgA-) to dominant genera (>1% of 479
total) of the gut microbiota at 1 month (nHealthy=27; nAllergic=19; nAsthma=10) and 12 months (nHealthy=28;
480
nAllergic=20; nAsthma=10) of age when comparing healthy children and children developing allergic (A, B)
481
and asthmatic symptoms (C, D). For a given genera, the value of the IgA index can range from positive 482
values, reflecting genera found dominantly in the IgA+ fraction, to negative values (genera found 483
dominantly in the IgA- fraction), as a measure of the degree of mucosal immune responsiveness to the 484
microbiota. LEfSe (Linear discriminant analysis Effect Size) algorithm, emphasizing both statistical 485
and biological relevance, was used for biomarker discovery. Threshold for the logarithmic discriminant 486
analysis (LDA) score was 2. Means with standard errors are indicated. * p<0.05; ** p<0.01. 487
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Fig. 5. IgA recognition patterns of the gut microbiota at 1 (left panels) and 12 months (right
488
panels). Principal component analysis (PCA) based on the IgA index (reflecting the differences in IgA
489
status, e.g. the ratio of IgA+ and IgA-) of the dominant genera (>1% of total) of the gut microbiota at 1 490
month (nHealthy=27; nAllergic=19; nAsthma=9; A, C) and 12 months (nHealthy=27; nAllergic=19; nAsthma=10; B,
491
D) of age when comparing healthy children with children developing allergic manifestations (A, B) or
492
with children developing asthmatic symptoms (C, D). 493
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Table I. Descriptive data of children included in this study.
495 Children Healthy % (no) Developing allergy % (no) P value* Developing asthma % (no) P value* Girls 46 (13) 40 (8) 0.66 40 (4) 1.00 Older siblings 57 (16) 50 (10) 0.62 50 (5) 0.70 Caesarean delivery 14 (4) 20 (4) 0.70 20 (2) 0.64 Furred pets 4 (1) 15 (3) 0.29 10 (1) 0.46 Maternal atopy 82 (23) 80 (16) 1.00 80 (8) 1.00 Breast-feeding (1 to 12 mo) 29 (8) 20 (4) 0.74 10 (1) 0.40 Antibiotic treatment (1-12 mo) 36 (10) 30 (6) 0.68 50 (5) 0.43 Day care (1-12 mo) 14 (4) 10 (2) 1.00 (0) 0.56 Probiotic group 54 (15) 60 (12) 0.66 60 (6) 1.00
*The x2 test was used to detect potential differences in frequencies between groups, except when the expected
496
frequency for any cell was less than 5, when the Fisher exact test was used.
497 498
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Stoolsamples
collected at 1 and 12 months of age
Cell Fixa on on Formaldehyde
Fluorescent Labeling An -IgA Labeling IgA+ IgA- DNA extrac on PCR 16S rDNA Pyrosequencing 454 Titanium Roche qPCR with universal bacterial primers ELISA assay IgA Total bacterial load Total IgA levels Gut microbial composi on Healthy children n=28 Children developing allergic symptoms at age 7 n=20 DNA extrac on The propor on of IgA-coated bacteria
Fig. 1. Schematic workflow of performed experiments. The proportion of the gut microbiota bound
to IgA (IgA+) or not (IgA-), in infants, was analyzed by flow cytometry-based sorting of fecal samples prior to 16S rDNA 454-pyrosequencing. In addition, total secretory IgA levels were estimated by ELISA tests, and the bacterial density measured using universal primers targeting the single-copy bacterial gene FusA.
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0 00 0 50 50 50 50 100 100 100 100 1 month 12 months * * * ** Healthy Allergic A A A A % I g A b o u n d 0 50 100 Healthy Asthmatic disease 1 month 12 months * * % I g A b o u n d B B B BFig. 2. Proportions of IgA-coated fecal bacteria in early infancy. A) The proportion of fecal bacteria
bound to IgA at 1 month and 12 months of age in children staying healthy (n=28, circles) or developing allergic symptoms (n=20, triangles) during the first 7 years of life. B) The proportion of fecal bacteria bound to IgA at 1 month and 12 months of age in children staying healthy (n=28, circles) or developing asthma (n=10, triangles) during the first 7 years of life. Median and interquartile ranges are indicated. *p < 0.05; **p < 0.01 (Mann–Whitney U-test).
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Fig. 3. Bacterial load and total fecal secretory IgA levels in healthy infants and infants developing allergic manifestations. A) The quantification of bacterial numbers was obtained by qPCR- detection
with universal primers targeting the gene FusA (present in single-copy in bacterial cells) and normalized by the number of human cells, determined by qPCR- detection with primers for the human
β-actin gene. nHealthy=28; nAllergic=20. B) Total secretory IgA levels in stool samples were measured
using ELISA immunoassay. 1 month of age: nHealthy=25; nAllergic=19; 12 months of age: nHealthy=27;
nAllergic=19. Means with standard errors are indicated. * p<0.05; ** p<0.01; *** p<0.001 (Mann
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dominant genera (>1% of total) of the gut microbiota at 1 month (nHealthy=27; nAllergic=19; nAsthma=10) and 12 months (nHealthy=28; nAllergic=20; nAsthma=10) of age when
comparing healthy children and children developing allergic (A, B) and asthmatic symptoms (C, D). For a given genera, the value of the IgA index can range from positive values, reflecting genera found dominantly in the IgA+ fraction, to negative values (genera found dominantly in the IgA- fraction), as a measure of the degree of mucosal immune responsiveness to the microbiota. LEfSe (Linear discriminant analysis Effect Size) algorithm, emphasizing both statistical and biological relevance, was used for biomarker discovery. Threshold for the logarithmic discriminant analysis (LDA) score was 2. Means with standard errors are indicated. * p<0.05; ** p<0.01.
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Fig. 5. IgA recognition patterns of the gut microbiota at 1 (left panels) and 12 months (right panels). Principal component analysis (PCA) based on the IgA index (reflecting the differences in IgA
status, e.g. the ratio of IgA+ and IgA-) of the dominant genera (>1% of total) of the gut microbiota at 1 month (nHealthy=27; nAllergic=19; nAsthma=9; A, C) and 12 months (nHealthy=27; nAllergic=19; nAsthma=10; B,
D) of age when comparing healthy children with children developing allergic manifestations (A, B) or
