It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate population of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) by a combination of collagenase-based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project.

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.

T-helper-17 (Th17) cells are a subset of CD4+ T cells that can produce the cytokine interleukin (IL)-17 and play vital roles in protecting the host from bacterial and fungal infections, especially at the mucosal surface. These are abundant in the small intestinal lamina propria (SILP) and their differentiation are associated with the colonization of the intestinal flora. Segmented filamentous bacteria (SFB) drew the attention of researchers due to their unique ability to drive the accumulation of Th17 cells in the SI LP of mice. Recent work has highlighted that SFB used microbial adhesion-triggered endocytosis (MATE) to transfer SFB antigenic proteins into small intestinal epithelial cells (SI ECs) and modulate host immune homeostasis. However, which components of SFB are involved in this immune response process remains unclear. Here, we examined the roles of SFB flagellins in Th17 cells induction using various techniques, including ELISA, ELISPOT, and RNA-seq in vitro and in vivo. The results show that the immune function of SFB flagellins is similar to SFB, i.e., induces the appearance of CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the SI LP. Furthermore, treatment of mice with SFB flagellins lead to a significant increase in the expression of genes associated with the IL-17 signaling pathway, such as IL-6, IL-1β, TNF-α, IL-17A, IL-17F, and IL-22. In addition, SFB flagellins have an intimate relationship with intestinal epithelial cells, influencing the expression of epithelial cell-specific genes such as Nos2, Duox2, Duoxa2, SAA3, Tat, and Lcn2. Thus, we propose that SFB flagellins play a significant role in the involvement of SFB in the induction of intestinal Th17 cells.

No abstract