Oropharyngeal microbiota composition and diversity
Eleven bacterial phyla were detected in the oropharyngeal microbiome of newly-arrived and long-term Chinese immigrants (Figure 2 A, Table E1). Firmicutes was dominant in both groups, comprising 54.3% and 53.4% of sequences in newly-arrived and long-term Chinese immigrants, respectively, followed by Actinobacteria, and Proteobacteria. At the genus level (Supplementary Figure 1, Table E2) there were 20 genera that account for 89.7% and 88.1% of the two groups respectively, with a minimum relative abundance of ≥1.0%. The relative abundance of Fusobacteria in newly-arrived immigrants (2.6%) was significantly lower than that of long-term immigrants (4.8%) (p< 0.01; FDR-corrected p= 0.066).
To further compare the microbial composition of long-term Chinese immigrants and newly-arrived Chinese immigrants, linear discriminant analysis effect size (LefSe) was used (Figure 2 B, threshold of LDA score: 2). The relative abundance of the genus Leptotrichia was higher among long-term immigrants, compared to that of newly-arrived Chinese immigrants. However, the abundance of genus Deinococcus was significantly lower in long-term immigrants Chinese immigrants. (Figure 2 B).
There was no significant difference in alpha diversity indices between the newly-arrived and long-term Chinese immigrants (Table E3 and Figure 2 C). The Bray-Curtis distance measurer was applied to examine differences in bacterial community composition and structure between the newly-arrived and long-term Chinese immigrants (Figure 2 D). No significant difference was observed in beta diversity according to the Bray-Curtis distance measure (ANOSIM, p= 0.431).
Oropharyngeal microbial taxa are significantly associated with immunological parameters
A Spearman correlation was used to test for the association of bacterial taxa with innate immune responses, adaptive immune responses and IgE levels. Figure 3 summarizes the results with a significance (p< 0.1, the p values have been adjusted for multiple comparison). In long-term Chinese immigrants, 25 taxa significantly correlated with at least one of these immunological parameters, namely, cytokine IL-6, IgG1 binding to bacterial antigens, and IgE, with majority of them showing negative correlations. In newly-arrived Chinese immigrant, seven taxa were significantly correlated with at least one immunological parameters, namely, IgG1 binding to virus antigens and IgE, with majority of them demonstrating positive correlations.
Interestingly, negative microbial taxa correlations with PC1s of cytokine IL-6 were found exclusively significant among 11 taxa in long-term immigrants (rho range: -0.60 ~ -0.72). Further, Spearman correlation tests were also conducted between these microbial taxa and cytokine IL-6 stimulated by six TLR ligands (rho range: -0.46 ~ -0.73, Figure 3 A). The taxa Lachnospiraceae_uncultured (Firmicutes), Erysipelotrichaceae UCG-007 (Firmicutes), Veillonella (Firmicutes), and Actinomycetales_ambiguous taxa (Actinobacteria) were negatively correlated with IL-6 stimulated by TLR ligands of Poly I:C, R848 and/or LPS.
In long-term Chinese immigrants the genera Oribacterium (Firmicutes), Neisseria (Proteobacteria) and Porphyromonas (Bacteroidetes) had a negative correlation with IgG1 antibody responses to bacterial antigen P6 or PspC, whereas Prevotella 7 (Bacteroidetes) was positively correlated with P6 (Figure 3 A). In newly-arrived Chinese immigrants the phylum Bacteroidetes was positively correlated with viral specific antigen to RV-A, but the order Actinomycetales (Actinobacteria) was negatively correlated with viral total antigen to RV-C (Figure 3 B).
A total of 12 taxa were significantly negatively correlated with total IgE or specific IgE to dog dander, while only the genus Neisseria was positively correlated with mould extract in long-term immigrants. The genus Saccharibacteria_Ambiguous was negatively correlated with total IgE in long-term immigrants, but the phylum Saccharibacteria was positively associated with total IgE, specific IgE for grass pollen mix and rye grass in newly-arrived immigrants (Figure 3).
Different microbial taxonomies and cytokine correlation comparison between long-term and newly-arrived Chinese immigrants
The paired-sample t test was used to investigate the correlation strengths between these bacterial taxa and the overall innate immune response (represented by the 138 cytokine measurements) in newly-arrived and long-term Chinese immigrants, respectively (Table E4). Correlation strength of bacteria with overall innate response was significantly different between newly-arrived and long-term Chinese immigrants after adjusting for multiple comparisons. Correlation trends for the genera Gemella (Firmicutes), Neisseria (Proteobacteria) and Kingella (Proteobacteria) were positive in newly-arrived Chinese immigrants, but negatively associated with long-term immigrants. Conversely, Prevotella 7 (Bacteroidetes) and Saccharibacteria_Ambiguous (Saccharibacteria) had negative correlation with the innate-immunity of newly-arrived Chinese immigrants, but positive correlation with that of long-term immigrants (Figure 4).