Dysbiosis gut microbiota associated with inflammation and impaired mucosal immune function in intestine of humans with non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) has recently been considered to be under the influence of the gut microbiota, which might exert toxic effects on the human host after intestinal absorption and delivery to the liver via the portal vein. In this study, the composition of the gut microbiota in NAFLD patients and healthy subjects was determined via 16S ribosomal RNA Illumina next-generation sequencing. Among those taxa displaying greater than 0.1% average abundance in all samples, five genera, including Alistipes and Prevotella, were significantly more abundant in the gut microbiota of healthy subjects compared to NAFLD patients. Alternatively, Escherichia, Anaerobacter, Lactobacillus and Streptococcus were increased in the gut microbiota of NAFLD patients compared to healthy subjects. In addition, decreased numbers of CD4+ and CD8+ T lymphocytes and increased levels of TNF-α, IL-6 and IFN-γ were detected in the NAFLD group compared to the healthy group. Furthermore, irregularly arranged microvilli and widened tight junctions were observed in the gut mucosa of the NAFLD patients via transmission electron microscopy. We postulate that aside from dysbiosis of the gut microbiota, gut microbiota-mediated inflammation of the intestinal mucosa and the related impairment in mucosal immune function play an important role in the pathogenesis of NAFLD.

Figure 1. Estimates of bacterial diversity as assessed by the Shannon index.

Figure 2. The overall structure of the gut microbiota of all samples.

The PLS-DA score plots were based on the relative abundance of the genera displaying greater than 0.1% average abundance. The green circles represent the 53 NAFLD patients, and the red circles represent the 32 healthy subjects.

Figure 3. Comparison of the relative abundance at different taxonomic levels between the 53 NAFLD patients and the 32 healthy subjects.

(A) Average phylum distribution (B) Primary genus composition in the two groups. *P < 0.05 and **P < 0.01.

Figure 4. Ultrastructural observation of the tight junctions in the duodenal mucosa (transmission electron microscopy, 43,000×).

(A) Mucosa from a NAFLD patient. (B) Mucosa from a healthy subject. (C) The width of the tight junction gap in 26 NAFLD patients and 10 healthy subjects. *P < 0.05 and **P < 0.01.

Figure 5. Immunohistochemical staining for the occludin protein in the duodenal mucosa (light microscopy, 100×).

The proportion of the occludin expression-positive area was scored as follows: 0 (none), 1 (1–25%), 2 (26%–50%), 3 (51%–75%), and 4 (≧76%). The staining intensity was graded according to the following criteria: 0 (no staining), 1 (weak staining, yellow), 2 (moderate staining, brown), and 3 (strong staining, deep brown). (A) Mucosa from a NAFLD patient. (B) Mucosa from a healthy individual. (C) The expression of the occludin protein in the duodenal mucosa in 29 NAFLD patients and 25 healthy subjects. *P < 0.05 and **P < 0.01.

Figure 6. Immunohistochemical staining for CD4+ and CD8+ T lymphocytes in the lamina propria of the duodenal mucosa.

(A) CD4+ T lymphocytes in mucosa from a NAFLD patient. (B) CD4+ T lymphocytes in mucosa from a healthy subject. (C) CD8+ T lymphocytes in mucosa from a NAFLD patient. (D) CD8+ T lymphocytes in mucosa from a healthy subject. (E) The number of CD4+ and CD8+ T lymphocytes in the lamina propria of the duodenal mucosa in 29 NAFLD patients and 25 healthy subjects. *P < 0.05 and **P < 0.01.

Figure 7. Relative mRNA expression levels of TNF-α, IL-6 and IFN-γ in 28 NAFLD patients and 27 healthy subjects. *P < 0.05 and **P < 0.01.