Presence of Segmented Filamentous Bacteria in Human Children and Its Potential Role in the Modulation of Human Gut Immunity

Abstract

Segmented filamentous bacteria (SFB) are commensal organisms that grow by anchoring a specialized holdfast structure to the intestinal walls of a variety of animals. Interaction of SFB with Peyer's patches in mice promotes the post-natal maturation of the immune system. We previously reported that the colonization of SFB in humans mainly occurs by 36 months of age, and is difficult to be detected afterward. In this study, we measured the level of SFB in intestinal fluids of human children. SFB were found via qPCR to represent a small fraction of the whole SFB-positive microbiota (10⁵ SFB in 10¹¹ total bacteria). Bacteria with filamentous segmented morphology were observed in intestinal fluids via fluorescent in situ hybridization, and from gut biopsies via scanning electron microscopy. SFB-specific DNA and peptide fragments were also identified via multiple displacement amplification PCR and mass spectrometry. There was an overall positive correlation between the presence of SFB and the titer of total secretory immunoglobulin A (sIgA), which is more apparent in intestinal fluids of the age group of 8-36 months. Afterward there was a decline of SFB in numbers correlated with a reduction of total sIgA. RT-qPCR analysis of the terminal ileal biopsies revealed that the expression of Th17 pathway genes were induced in SFB-positive samples, while the markers of T and B cell receptor signaling pathways were also upregulated. Collectively, these data suggest that SFB is a rare member of microbiota, and may play an important role in the development of human gut immunity.

Keywords: SFB; SIgA; Th17 cells; ileum microbiota; immunity.

FIGURE 1

Quantification of SFB abundance in luminal fluids from the terminal ilea of the age group of 8- to 48-month-old of Chinese children. The luminal fluids of disease-free human subjects (n–24) as shown in Supplementary Table S1 (qPCR column) obtained via standard endoscopic procedure were used for the extraction of microbiota DNA. 100 ng amount of DNA per sample was used for qPCR amplification using SFB-specific primers and universal bacterial primers for total bacteria as described in the Section “Materials and Methods.” Comparison of the level of SFB vs. total bacteria.

FIGURE 2

SEM and FISH micrographs of human ileal biopsies. The specimens were processed for the observation of microbiota by SEM and FISH as described in the Section “Materials and Methods.” Samples processed for SEM were labeled in the SEM column in Supplementary Table S1. Terminal ilea of patient 4 (a,b) observed under SEM with magnification of 2,300 and 470, respectively. SFB-like structures as visualized with DAPI staining (c,e) and hybridization with SFB-specific probe (d,f) of patient 4 and 20 (long filamentous shape) observed under epi-fluorescent microscope with magnification of 1,000; SFB by DAPI staining (g) and hybridization with SFB-specific probe (h) of patient 22 (short rod shape).

FIGURE 3

(A) Overall relationship between the level of sIgA in luminal fluids from the SFB-positive and SFB-negative patients. (B) Relationship between the level of sIgA and the ages of children. A total number of 47 samples (23 SFB-positive and 24 SFB-negative) were used. 500 μl of luminal fluids from each patient were used for the measurement of sIgA as described in the Section “Materials and Methods.” The supernatants were assayed for total secretory IgA using a human IgA ELISA Detection Kit as described in the section “Materials and Methods.” The sIgA was expressed as μg/ml of fecal materials. P-values were calculated using Student’s t-test.

FIGURE 4

Effect of SFB colonization on Th17 pathway gene expression in human terminal ilea specimens. The tissue biopsies of the terminal ilea from a total of 22 human subjects (11 SFB-positive and SFB-negative, Supplementary Table S1) were used for the extraction of mRNA. 500 ng amount of mRNA was used for RT-qPCR with primers for the Th17 pathway markers as indicated in Supplementary Table S2. (A,B) are a select group of Th17 pathway genes used for analysis.

FIGURE 5

Effect of SFB colonization on the induction of T and B cell receptor signaling pathways in human terminal ilea biopsies. The 22 biopsies were collected and used for the analysis of the gene expression of a select group of receptor pathway genes. Data are expressed as the mean ± SD (n = 22). P-values were calculated using the Student’s t-test. RT-qPCR was performed as described in the Supplementary Materials and Methods. (A,B) are a select group of BCR and TCR signaling pathway genes, respectively.