Allergy‐related diseases and early gut fungal and bacterial microbiota abundances in children

Abstract Background The early gut microbiota has been proposed as an important link between environmental exposures and development of allergy‐related diseases. Beyond the widely investigated associations between the gut bacterial microbiota, we investigated the involvement of early gut mycobiota and gut permeability in the pathogenesis of asthma, allergic rhinoconjunctivitis (AR) and eczema. Methods In the Probiotics in the Prevention of Allergy among Children in Trondheim trial with maternal probiotic supplementation, we collected faecal samples at four timepoints between 0 and 2 years from a cohort of 278 children. Clinical information on allergy‐related diseases was collected in a paediatric examination at 2 years and questionnaires at 6 weeks and 1, 2 and 6 years. By quantitative PCR and 16S/ITS1 MiSeq rRNA gene sequencing, we analysed the gut bacterial and fungal microbiota abundance and bacterial diversity and explored associations with allergy‐related diseases. We also measured gut permeability markers (lipopolysaccharide‐binding protein [LBP] and fatty acid‐binding protein 2 [FABP2]). Results Children with higher fungal abundance at 2 years were more likely to develop asthma and AR by 6 years, odds ratios 1.70 (95% CI: 1.06–2.75) and 1.41 (1.03–1.93), respectively. We explored causal connections, and children with eczema at 1–2 years appeared to have more mature bacterial microbiota, as well as being depleted of Enterococcus genus. Although LBP and FABP2 did not correlate with eczema, increased bacterial abundance was associated with increased serum FABP2. Conclusions We observed positive associations between gut fungal abundance and allergy‐related disease, but increased gut permeability does not appear to be involved in the underlying mechanisms for this association. Our findings should be confirmed in future microbiota studies.


| BACKGROUND
Allergy-related diseases, such as asthma, allergic rhinoconjunctivitis (AR) and eczema, are chronic inflammatory diseases. The prevalence of these diseases has increased over the last decades and collectively they are now the most prevalent chronic diseases in childhood. 1 Environmental factors play an important role in the rising prevalence given the rapidity of its increase, and changing microbial exposures are suspected to be one of the driving factors. 2 The so-called hygiene hypothesis originally postulated that reduced childhood infections may predispose to allergy-related diseases; however, this hypothesis has evolved to place a greater emphasis on a depletion or perturbation of the early commensal microbiome and the influence of this disturbance on the developing immune system. 3,4 Epidemiological studies have identified a number of environmental risk factors for allergy-related diseases, such as delivery by Caesarean section, formula-feeding, urban living, less animal contact and Western diet. 5,6 The influence of each of these factors on allergyrelated disease may in part be due to reduced quantity or diversity of microbial exposures in early life, and through altered development of the gut microbiota in particular. The gut represents a major site where the immune system interacts with vast quantities of microbes.
Early low bacterial diversity and varying microbial patterns have been shown to be associated with development of allergy-related diseases later. [7][8][9] Whilet the gut microbiota mainly consists of bacteria, fungi also colonise the gut.
The gut mycobiota is considered to be a fundamental constituent of the gut microbiota, contributing to around 1% of the microbial cells in the gut. 10 However, our understanding of the role of mycobiota in allergy-related disease is scarce, and more knowledge is called for. 11 The association between gut mycobiota and allergy appears to be more nuanced than for bacteria. Growing up in rural environments with higher fungal content may decrease the risk of allergy-related diseases, 6,12 whereas indoor fungal exposure through moulds has been associated with an increased risk. 12 Two recent small studies have found associations between neonatal fungal dysbiosis and asthma or wheezing in pre-school age. 8,13 Furthermore, murine research on fungi has shown that gavage feeding of certain fungi (and possibly perturbing the gut mycobiota) can induce allergic airway disease in mice. 14 The primary point of contact for both bacterial and fungal members of the gut microbiota and their host is the gut mucosa. The gut mucosal immune system is optimally adapted to tolerate commensal microbes in the gut lumen, while expelling pathogenic invasive microbes by activating proinflammatory reactions, as well as cell wall or intracellular pathogen recognition receptors of epithelial cells. 15 As such, a more permeable gut mucosa, or leaky gut, may allow microbes to penetrate the intestinal epithelial lining more readily and lead to an increased inflammatory response to the gut microbiota. The gut permeability in infancy could be one of the mechanisms by which the gut microbiota is involved in the development of allergy-related diseases, as higher gut permeability has been observed in both adults and children (5-12 years) with asthma, 16,17 and eczema. 18,19 Two such markers of gut permeability are lipopolysaccharide-binding protein (LBP) and fatty acid-binding protein 2 (FABP2, also called intestinal-type FABP, or FABPI). LBP is part of the innate immune response that opsonises lipopolysaccharides (LPS or endotoxin, a cell wall constituent of Gram-negative bacteria) when this crosses the gut barrier and serves thus as a marker of metabolic endotoxaemia. 20 FABP2 is expressed in the gut from duodenum to caecum and is a marker of gut permeability and enterocyte turnover or damage. 21 Higher circulating levels of both markers are observed in neonatal necrotising enterocolitis. 22 Several findings indicate that the gut microbes are associated with allergy development. However, the potential association between early gut fungal and bacterial microbiota, and development of allergy-related diseases is largely unknown. The aim of this study was, therefore, to explore the associations between the early gut fungal and bacterial microbiota and gut permeability during the first 2 years of life, and the development of allergy-related diseases up to 6 years of age in a prospective cohort.

| Materials
The faecal samples in this study were collected during the Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT) study, a Norwegian randomised placebo-controlled trial investigating the use of maternal probiotic supplementation in the prevention of eczema and other allergy-related diseases. 23 Briefly, mothers were randomised to drink either sterile placebo milk or probiotic milk containing three probiotic bacteria (5 � 10 10 colony-forming units   Figure 1). The mothers included in the current analysis were 8 months older and of longer education and their children had received more antibiotics between 1 and 2 years of age; other than that the groups were comparable. 25 These children provided in total 1015 faecal samples at up to four timepoints (10 days, 3 months, 1 and 2 years). The parents collected the samples from the diaper into a Cary-Blair transport medium (about 20 times dilution) with an enclosed spoon, and they were asked to place the samples in a −18°C freezer immediately before transport to the laboratory and storage at −80°C.

| Follow-ups and definitions of allergy-related diseases
The parents were asked to complete questionnaires on home environment of the child at 6 weeks after birth, 1 year and 2 years, and child health questionnaires at 1, 2 and 6 years. Additionally, a clinical evaluation of allergy-related diseases was performed by a paediatrician at 2 years of age and by research assistants at 6 years of age.
Current eczema (within the last year) at 2 or 6 years was defined according to The U.K. Working Party's diagnostic criteria for atopic eczema. 26,27 Ever eczema at 2 or 6 years was defined using the questionnaire responses if the parents reported that the child had 'ever had eczema' and 'ever had a recurring itchy rash during 6 months'. Similarly, asthma and AR were defined using the questionnaire if the parents reported that the child was 'ever diagnosed with asthma by a physician' for asthma and 'ever had hay-fever, allergic rhinitis or allergic conjunctivitis' for AR.

| Microbiota analyses
The microbiota analyses were fully explained in a previous article. 25 The faecal samples were homogenised before DNA extraction by a Months of breastfeeding, months, mean (SD) 11.0 (4.6) 260 Antibiotics administration, n (%) F I G U R E 1 Causality analysis. Overview of how the causality analysis between fungal and bacterial abundances and eczema was performed. Linear regressions were used to estimate if eczema diagnosed at or prior to each timepoint predicted fungal or bacterial abundance at that timepoint. Logistic regressions were used to estimate if fungal or bacterial abundance at each timepoint predicted eczema up to 2 years (6 years for the lowest analysis) among those with no history of eczema by that timepoint. The included participants in this analysis were those who could develop eczema at 0-6 years of age. This number adds up to n = 212, as three participants did not provide the presentation age of eczema in the questionnaires SCHEI ET AL.
bacterial protocol, 28 since there were no validated protocols at the time for fungal DNA extraction. The microbial abundances were quantified by quantitative PCR with bacteria-targeted primers (V3-V4 part of the 16S rRNA gene), 29 and fungi-targeted primers (ITS1 part of 18S rRNA gene). 30 Out of the 1015 samples, 999 (98%) and 653 (64%) had quantifiable levels of bacteria and fungi, respectively (Supplementary Table S1A). 25 The ITS1 qPCR cut-off value was set to the negative control if fungal abundance was lower than negative control. were not sequenced due to low total quantities, and those with less than 6000 reads per sample were excluded after sequencing due to insufficient data. This resulted in that 37 (4%) samples were sequenced for fungi. For this reason, only bacterial diversity is used in the analysis.
For annotation, Greengenes Database was used for bacteria, and a self-curated concordance system was used for fungi as there were no well-established methods for fungal annotation. 33

| Gut permeability markers
We analysed serum levels of LBP and FABP2. LBP was measured by ELISA (Human LBP, Merck, own positive controls) and FABP2 by ELISA (Human FABP2/I-FABP Quantikine ELISA Kit, R&D Systems, producer's positive controls), following the producers' protocols, including negative controls on all plates.

| Statistics
The data from both the probiotic and placebo arm of the ProPACT trial are used in the current analysis. Previous analysis of the infant stool samples has indicated that only LGG appears to be transferred from mother to child. Although we observed greater abundance and presence of LGG at 10 days and 3 months in infant stool samples from the probiotics group, the relative abundance of LGG was still low and did not persist at 1 or 2 years of age. 23 Furthermore, maternal probiotic supplementation was not associated with any change in the overall microbial composition of diversity at any age, 23 or with fungal abundance. 33  ANCOM-BC was used for longitudinal taxonomic statistic. 36 Estimated associations are presented with 95% confidence intervals.

| Fungal and bacterial abundance and allergyrelated diseases
We found positive associations between the cumulative incidence of ever asthma and ever AR at 6 years and higher faecal fungal On the other hand, when we considered the association between an existing eczema diagnosis and fungal abundance at each timepoint, we found that eczema debut before 3 months, 1 or 2 years was not conclusively associated with fungal abundance at these timepoints (Supplementary Figure S4 and Supplementary Table S5).
Regarding bacterial abundance and allergy-related diseases, we found no clear associations in equivalent analyses (Supplementary Table S5).

| Microbial taxa and eczema
Principal coordinate analysis (PCoA) ordination plots indicated that the fungal and bacterial variance of the microbiota is partially determined by age ( Figure 3A,B and Supplementary Figure S5 A LEfSe analysis of the fungal and bacterial microbiota was performed for different age groups to explore if specific taxa correlated with fungal abundance and eczema. Specific taxa were found significantly over-and underrepresented in children with and without ever eczema (Figure 4), and in children with high and low fungal abundance ( Figure 4). In particular, we found high abundances of Enterococcus sp. at 2 years in children without eczema. Several taxa were also under-and over-represented with age and fungal abundance (Supplementary Figure S7A,B). Longitudinal ANCOM-BC provided no statistically significant associations with allergy-related diseases. There were neither statistically significant associations between LBP and eczema, nor between LBP and fungal abundance (Supplementary Table S8). Increased bacterial diversity was associated with higher LBP at 3 months (β 0.32, 95% CI: 0.05-0.59), but with lower LBP at 2 years (β −0.22, 95% CI −0.43 to −0.01).

| DISCUSSION
In this population cohort following children from birth to 6 years, we found that children with higher fungal abundance at 2 years of age were more likely to have allergy-related diseases within the first 6 years of life. In an attempt to identify the causal direction of this associations, subgroup analyses suggested that higher fungal abundance at 2 years may precede later eczema development. However, this should be interpreted cautiously because of the uncertainty around this estimate. We also observed that early bacterial microbiota succession might appear prior to eczema, however markers of intestinal permeability were not consistently associated with eczema, fungal or bacterial abundance, or bacterial diversity.
We found that fungal abundance was associated with allergyrelated diseases. This is in line with a small case-control study with 20 Ecuadorian neonates, 13 where higher fungal abundance was associated with allergy-related diseases. Although the analysis between fungal abundance and allergy-related diseases was not constructed to prove causality, our causality analysis could imply that fungal abundance was present prior to development of allergyrelated diseases. Fungi are eukaryotes that are evolutionarily closer to humans than bacteria, sharing a great number of common biochemical pathways. Thus, they could impact human pathways through production of human-like substances. Fungi are well-known producers of prostaglandins and leukotrienes. 37 The Candida yeast, and likely other fungi, can produce prostaglandins that may reduce gut macrophage activity against fungi and thereby improve fungal gut colonisation. 38 By orchestrating the immune system through self-produced priming cytokines, fungi could in theory induce the native T helper (T H ) cells to pursue a T H 2 direction, which could create both a fungus-friendly gut environment and a predisposition to allergy-related disease development. 39 Whilst this is a plausible biological explanation for a causal association between gut mycobiota and later allergy-related diseases, our results cannot exclude that the association could also be driven by the mechanisms working in the opposite direction. Allergy-related diseases and their underlying immunopathology could themselves promote a fungus-friendly gut environment.
Our taxonomic analyses suggested that 1-year-olds were prone to develop eczema if their bacterial microbiota compositions more closely resembled the composition seen in 2-year-olds. Neither delivery, antibiotics administration nor breastfeeding appeared to explain the taxonomic succession from 1 to 2 years. The absence of the lactic-acid bacteria Enterococcus spp. at 2 years was associated with eczema at 2 years. Enterococcus depletion has also been found in allergic 8-year-old Swedish children. 40 Some enterococci are also used as probiotics. 41  -7 of 10 abundance. As none of these markers were associated with eczema, the connection between fungal abundance and eczema does probably not involve increased gut permeability and metabolic endotoxaemia.
However, the finding could indicate that bacterial abundance might affect the gut wall permeability. Different methods and younger participants in our study could explain why we could not replicate the gut permeability-allergy association found in older and smaller studies.

| Limitations
Whilst fungal abundance was quantifiable for approximately two thirds of the stool samples, only a minority of these could be sequenced and we therefore have limited opportunity to investigate the role of the mycobiota composition on allergy-related disease.
Also, the number of repeats of the fungal ITS gene sequence varies between fungal species, which could increase the random error in the analysis. A fungal DNA extraction technique would be preferred if such were available, to improve quantification and sequencing. 32

| Strengths
In this prospective cohort study of children from a general population followed for 6 years, we pursued an explorative approach with the intention to reveal mechanisms underlying the associations between both the fungal and bacterial microbiota and allergy-related diseases.
A large sample size and up to four consecutive samples at different ages were analysed. Furthermore, we have quantified the total fungal and bacterial abundance and included markers of gut permeability to explore potential causal pathways of these findings.

| CONCLUSIONS
To conclude, our study showed that gut fungal abundance at 2 years was associated with the development of allergy-related diseases in the first 6 years of life. Whilst we could not conclusively determine the causal direction of this association, this study indicated that the fungal and bacterial microbiota may both contribute to the complex pathogenesis of allergy-related disease. To approach a pathophysiologic mechanism, further scientific efforts should be put into larger infancy cohorts with even more frequent early measurements and improved methods of gut fungal and bacterial microbiota and allergyrelated diseases.

ACKNOWLEDGEMENTS
Our greatest thanks to all participating families, as well as all GPs and community nurses who made this study possible. The assistance from Inga Leena Angell (MS) at the Norwegian University of Life Sciences was most appreciated.
The study was supported by the Research Council of Norway and internal funds from NTNU-Norwegian University of Science and Technology. The probiotic intervention material was provided by TINE BA. The funders had no role in the study design, the data management or the review and publishing process. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

CONFLICT OF INTEREST
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AUTHORS' CONTRIBUTIONS
Kasper Schei did the microbiome data generation, the statistical analysis, interpretation and drafted the initial manuscript; Melanie Rae Simpson contributed to the concept, statistical analysis and interpretation and reviewed and revised the manuscript; Knut Rudi was involved in the microbiome data generation and reviewed and revised the manuscript; Torbjørn Øien designed the study, enrolled the participants, coordinated and supervised the data collection and reviewed and revised the manuscript; SS aided in the conceptualisation and design of the study and reviewed and revised the manuscript; Rønnaug Astri Ødegård supervised the study, conceptualised and designed the study, interpreted the data and reviewed and revised the manuscript. All authors approved of the final manuscript as submitted and agree to be accountable for all aspects of the work.