Inhibition of Sphingosine-1-Phosphate-Induced Th17 Cells Ameliorates Alcohol-Associated Steatohepatitis in Mice

Abstract

Background and aims: Chronic alcohol consumption is accompanied by intestinal inflammation. However, little is known about how alterations to the intestinal immune system and sphingolipids contribute to the pathogenesis of alcohol-associated liver disease (ALD).

Approach and results: We used wild-type mice, retinoid-related orphan receptor gamma t (RORγt)-deficient mice, sphingosine kinase-deficient mice, and local gut anti-inflammatory, 5-aminosalicyclic acid-treated mice in a chronic-binge ethanol feeding model. Targeted lipidomics assessed the sphingolipids in gut and liver samples. Gut immune cell populations, the amounts of sphingolipids, and the level of liver injury were examined. Alcohol intake induces a pro-inflammatory shift in immune cell populations in the gut, including an increase in Th17 cells. Using RORγt-deficient mice, we found that Th17 cells are required for alcohol-associated gut inflammation and the development of ALD. Treatment with 5-aminosalicyclic acid decreases alcohol-induced liver injury and reverses gut inflammation by the suppression of CD4⁺ /RORγt⁺ /interleukin-17A⁺ cells. Increased Th17 cells were due to up-regulation of sphingosine kinase 1 activity and RORγt activation. We found that S1P/S1PR1 signaling is required for the development of Th17 cell-mediated ALD. Importantly, in vivo intervention blocking of S1P/S1PR1 signaling markedly attenuated alcohol-induced liver inflammation, steatosis, and damage.

Conclusions: Gut inflammation is a functional alteration of immune cells in ALD. Reducing gut Th17 cells leads to reduced liver damage. S1P signaling was crucial in the pathogenesis of ALD in a Th17 cell-dependent manner. Furthermore, our findings suggest that compounds that reduce gut inflammation locally may represent a unique targeted approach in the treatment of ALD.

Figure 1. ALD is associated with a pro-inflammatory shift in intestinal immune cells.

WT mice (A-B) and RORγt^−/− (C-F) mice were fed control (pair-fed, PF) or alcohol (alcohol-fed, AF) diet and sacrificed 15–22 days later. (A) Intracellular staining of IL-17A and RORγt in CD4⁺ T cells in the lamina propia from large intestine (LI-LPL) or small intestine (SI-LPL). (B) ELISA analysis of IL-17A in the supernatant of colonic LPLs stimulated with IL-23 (left) and real-time PCR analysis of IL-17A and Rorc mRNA in colonic LPLs (right). (C-F) WT mice or RORγt^−/− mice were fed alcohol diet and sacrificed 15 days later. (C) HE and Oil red O staining of liver tissue. (D) Serum levels of ALT and AST and hepatic triglycerides. (E) Frequencies of immature myeloid cells (CD11b⁺Gr-1⁺) in the liver and Th1 and Tregs cells in the gut LPLs. (F) Large intestine was stained by H&E. Data in all panels are presented as Mean ± SEM. n=11 * P<0.05; ** P<0.01

Figure 2. 5-ASA administration protects mice from ALD.

C57BL/6 mice on a pair-fed or binge ethanol diet, with 5-ASA oral supplementation (50 mg/kg/day) or vehicle for 15 days (A-D) or for 28 days (E-G). (A) H&E and Oil red O staining of liver tissue. (B) Hepatic triglycerides. (C) Serum levels of ALT and AST. (D) Serum levels of lipids were analyzed by piccolo lipid pane plus. (E) Fibrosis was evaluated by Sirius Red staining. (F) Real-time PCR analysis of the expression of indicated genes in liver fibrosis. (G) Serum markers of liver fibrosis, including hyaluronic acid and TIMP-1. Data in all panels are presented as Mean ± SEM. n>15 * P<0.05; ** P<0.01

Figure 3. 5-ASA improves liver and gut inflammation in mice during alcohol feeding.

(A) Frequencies of immature myeloid cells (CD11b⁺Gr-1⁺) and macrophages (CD11b⁺F4/80⁺) in the liver. (B) Immunohistochemistry staining of myeloperoxidase (MPO) in the liver. (C) Real-time PCR analysis of the expression of indicated genes in the liver. (D) H&E staining of small and large intestine. (E) Alcian Blue staining and immunohistochemistry staining of mucin 2 of colonic tissue. (F) Plasma levels of Dextran-FITC for gut permeability. (G) Intracellular staining of IL-17A⁺ CD4⁺ T cells in LPL of colon or granzyme B⁺ γδ⁺ T cells in IEL of small intestine. (H) Real-time PCR analysis of the expression of indicated genes in the colonic LPL. Data in all panels are presented as Mean ± SEM. n>15 * P<0.05; ** P<0.01

Figure 4. Activation of sphingosine kinase and metabolism of sphingolipids in ALD.

(A) Real-time PCR analysis of the expression of sphingosine kinase in the colon, small intestine and liver. (B) Levels of S1P, DHS1P and acyl-chain ceramides in the liver. (C) Levels of sphingosine, sphinganine, acyl-chain ceramides and sphingomyelin in the gut. (D) Levels of acyl-chain ceramides and sphingomyelin in the serum of moderate, severe alcoholic hepatitis (AH) patients or healthy donors (normal liver). n=13 (E) Immunohistochemistry staining analysis of SK1 expression in the liver of AH patients and healthy control. (F) Levels of pStat3 were evaluated using immunoblotting and immunofluorescence in the colon of mice. Data from mice are presented as Mean ± SEM. n=8 * P<0.05; ** P<0.01

Figure 5. Deficiency of SK1 attenuates alcoholic liver inflammation, steatosis, and damage.

WT or SK1^−/− mice were fed alcohol diet and sacrificed 15 days later. (A) H&E staining and Oil red O staining of liver. (B) Serum ALT and AST. (C) Hepatic triglycerides. (D) Frequencies of immature myeloid cells, macrophages, Th1, and Th17 cells in the liver. (E) Immunohistochemistry staining of myeloperoxidase (MPO) in the liver. (F) Real-time PCR analysis of the relative expression of indicated genes in the liver. Data in all panels are presented as Mean ± SEM. n=11 * P<0.05; ** P<0.01

Figure 6. Deficiency of SK1 attenuates the gut inflammation and accumulation of Th17 cells in ALD.

(A) H&E staining of small and large intestine. (B) Alcian Blue staining and immunohistochemistry staining of mucin 2 in colonic tissue. (C) Plasma levels of Dextran-FITC for gut permeability. (D) Real-time PCR analysis of the expression of indicated genes in the colon (left) and IL-17A and Rorc mRNAs (right) in colonic LPLs. (E) ELISA analysis of IL-17A or IL-23 in the supernatant of colonic LPLs. (F) Intracellular staining of IL-17A⁺ and IFN-γ⁺ CD4⁺ T cells from gut LPL. (G) Relative levels of S1P in the ileum of alcohol-fed mice. (H) Immunofluorescence was used to evaluate the expression of pSTAT3 in the ileum. Data in all panels are presented as Mean ± SEM. n=11 * P<0.05; ** P<0.01

Figure 7. Deficiency of SK2 promotes liver injury in ALD.

WT or SK2^−/− mice on a chronic-binge ethanol diet, with/without 5-ASA oral supplementation (50 mg/kg/day) or vehicle for 15 days. (A) H&E staining and Oil red O staining of liver. (B) The levels of serum ALT, AST and hepatic triglycerides. (C) Frequencies of immature myeloid cells (CD11b⁺Gr-1⁺) in the liver. (D) H&E staining of small and large intestine. (E) Real-time PCR analysis of the levels of Muc2, Muc5a, IL-17A, Rorc and SK1 mRNA in the colon. (F) Intracellular staining of IL-17A, IFN-γ, Foxp3 or RORγt in CD4⁺ T cells from colonic LPL. Data in all panels are presented as Mean ± SEM. n=7 * P<0.05; ** P<0.01

Figure 8. Pharmacologic intervention via inhibition of S1P/SPR1 signaling ameliorates ALD progression.

(A) H&E staining and Oil red O staining of liver. (B) Serum levels of ALT and hepatic triglycerides. (C) Frequencies of immature myeloid cells (Top) and immunohistochemistry analysis of the expression of myeloperoxidase (MPO) (bottom) in the liver. (D) Real-time PCR analysis of the expression of indicated genes in the liver. (E) H&E staining of large intestine. (F) Real-time PCR analysis of the expression of indicated genes in the colon. (G) The proportion of IL-17A, IFN-γ, Foxp3 or RoRγt in CD4⁺ T cells from colonic LPL. Data in all panels are presented as Mean ± SEM. n=7 * P<0.05; ** P<0.01