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. 2023 Apr 21:14:1130184.
doi: 10.3389/fimmu.2023.1130184. eCollection 2023.

Modulating sphingosine 1-phosphate receptor signaling skews intrahepatic leukocytes and attenuates murine nonalcoholic steatohepatitis

Chieh-Yu Liao  1 , Fanta Barrow  2 , Nanditha Venkatesan  1 , Yasuhiko Nakao  1 , Amy S Mauer  1 , Gavin Fredrickson  2 , Myeong Jun Song  1   3 , Tejasav S Sehrawat  1 , Debanjali Dasgupta  1 , Rondell P Graham  4 , Xavier S Revelo  2 , Harmeet Malhi  1
Affiliations

Modulating sphingosine 1-phosphate receptor signaling skews intrahepatic leukocytes and attenuates murine nonalcoholic steatohepatitis

Chieh-Yu Liao et al. Front Immunol. .

Abstract

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid associated with nonalcoholic steatohepatitis (NASH). Immune cell-driven inflammation is a key determinant of NASH progression. Macrophages, monocytes, NK cells, T cells, NKT cells, and B cells variably express S1P receptors from a repertoire of 5 receptors termed S1P1 - S1P5. We have previously demonstrated that non-specific S1P receptor antagonism ameliorates NASH and attenuates hepatic macrophage accumulation. However, the effect of S1P receptor antagonism on additional immune cell populations in NASH remains unknown. We hypothesized that S1P receptor specific modulation may ameliorate NASH by altering leukocyte recruitment. A murine NASH model was established by dietary feeding of C57BL/6 male mice with a diet high in fructose, saturated fat, and cholesterol (FFC) for 24 weeks. In the last 4 weeks of dietary feeding, the mice received the S1P1,4,5 modulator Etrasimod or the S1P1 modulator Amiselimod, daily by oral gavage. Liver injury and inflammation were determined by histological and gene expression analyses. Intrahepatic leukocyte populations were analyzed by flow cytometry, immunohistochemistry, and mRNA expression. Alanine aminotransferase, a sensitive circulating marker for liver injury, was reduced in response to Etrasimod and Amiselimod treatment. Liver histology showed a reduction in inflammatory foci in Etrasimod-treated mice. Etrasimod treatment substantially altered the intrahepatic leukocyte populations through a reduction in the frequency of T cells, B cells, and NKT cells and a proportional increase in CD11b+ myeloid cells, polymorphonuclear cells, and double negative T cells in FFC-fed and control standard chow diet (CD)-fed mice. In contrast, FFC-fed Amiselimod-treated mice showed no changes in the frequencies of intrahepatic leukocytes. Consistent with the improvement in liver injury and inflammation, hepatic macrophage accumulation and the gene expression of proinflammatory markers such as Lgals3 and Mcp-1 were decreased in Etrasimod-treated FFC-fed mice. Etrasimod treated mouse livers demonstrated an increase in non-inflammatory (Marco) and lipid associated (Trem2) macrophage markers. Thus, S1P1,4,5 modulation by Etrasimod is more effective than S1P1 antagonism by Amiselimod, at the dose tested, in ameliorating NASH, likely due to the alteration of leukocyte trafficking and recruitment. Etrasimod treatment results in a substantial attenuation of liver injury and inflammation in murine NASH.

Keywords: Amiselimod; Etrasimod; fatty liver; lipotoxicity; sphingolipids.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Etrasimod treatment reduces liver injury. (A) Timeline of mouse study and analysis after euthanasia. Dietary feeding started 6 weeks after acquisition of C57BL/6J male mice at 12 weeks of age. Mice were randomized to receive standard CD or FFC diet for 24 weeks. At 20 weeks of feeding, CD and FFC cohorts received vehicle or Etrasimod and vehicle or Amiselimod drug treatment for 4 weeks. Mice were euthanized at the end of gavage therapy and processed for analysis. (B) Representative liver histology shown by Hematoxylin and Eosin (H&E) staining of vehicle or Etrasimod treated mice in CD and FFC cohorts. Scale bar equals 50 μm. (C) Steatosis component of the NAFLD Activity Score for mouse cohorts treated with vehicle or Etrasimod. Each mouse is graded for steatosis (0–3). Less than 5% steatosis = 0, 5~33% = 1, 34~66% = 2, >66% = 3. Each dot represents one biological replicate. (D) The inflammatory (0-3) component score in NAS grading is shown for CD and FFC cohorts treated with vehicle or Etrasimod. If there are no inflammatory foci = 0, <2 inflammatory foci = 1, 2~4 inflammatory foci = 2, >4 inflammatory foci = 3. (E) Plasma ALT levels for CD and FFC cohorts treated with vehicle or Etrasimod at completion of the study. CD (n=10) and FFC (n=10). *p<0.05, ***p<0.001.
Figure 2
Figure 2
Metabolic characterization of CD and FFC-fed mice with Etrasimod treatment. (A) Liver mass, (B) liver to body mass ratio, (C) and body mass of groups treated with vehicle or Etrasimod. (D) Total cholesterol and (E) fasting blood glucose with vehicle or Etrasimod treatment. (F) Area under the curve (AUC) for the glucose tolerance test at 22 weeks on diet and 2 weeks of Etrasimod treatment. CD (n=10) and FFC (n=10). *p<0.05, ***p<0.001.
Figure 3
Figure 3
Flow cytometry gating strategy. (A) Flow cytometry gating strategy that is used to isolate immune populations within the liver of CD-fed mice and (B) FFC-fed mice. Immune populations were identified as follows: NK cells (CD3-NK1.1+), NKT cells (CD3+NK1.1+), B cells (CD3-NK1.1-CD19+B220+), DCs (CD3-NK1.1-CD19-B220-CD11c+), PMNs (CD11b+Ly6G-), CD11b+ myeloid cells (CD11b+ Ly6G-). T cells (CD3+NK1.1-) were selected for further analysis to identify CD4 T cells (CD3+NK1.1-CD4+CD8a-), CD8 T cells (CD3+NK1.1-CD4-CD8a+) and DNT cells (CD3+NK1.1-CD4-CD8a-).
Figure 4
Figure 4
Etrasimod treatment alters intrahepatic leukocyte populations. (A) Number of CD45+ cells per gram acquired for FACS analysis in CD and FFC cohorts. (B) Subsets of CD45+ cells, (C) immune cell profiling expressed in %CD45 cells that are T cells, B cells and NKT cells by FACS analysis in livers from CD and FFC cohorts that received vehicle or Etrasimod treatment. (D) T-cell subtypes expressed in %CD45 cells that are CD4 positive, CD8 positive or double negative in CD and FFC cohorts. (E) Subsets of CD45+ cells that are NK cells, CD11b+ myeloid cells, PMNs and DCs. (F) Immune cells expressed in %CD45 that are NK cells, CD11b+ myeloid cells, PMNs and DCs. CD (vehicle n=4, Etrasimod n=6) and FFC (vehicle n=5, Etrasimod n=5). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 5
Figure 5
Macrophage accumulation and inflammatory markers are reduced in Etrasimod treated mice. (A) Representative images showing immunohistochemistry for Mac-2 in liver tissue sections for vehicle and Etrasimod treated mice in both CD and FFC cohorts. Scale bar equals 50 μm. (B) Mac-2 staining was quantified as percentage of positive immunoreactive area in CD and FFC-fed mice treated with vehicle or Etrasimod. (C–E) Relative mRNA expression of Cd68, Lgals3 and Ly6c in CD and FFC cohorts treated with vehicle or Etrasimod. (F–H) Relative mRNA expression of monocyte chemoattractant protein-1 (Mcp1), tumor necrosis factor alpha (Tnfa) and interleukin-1 beta (Il1b) in liver tissues of CD and FFC-fed mice treated with vehicle or Etrasimod. (I–K) Relative mRNA expression of Timd4, Marco and Trem2 in CD and FFC cohorts treated with vehicle or Etrasimod. CD (vehicle n=4, Etrasimod n=6) and FFC (vehicle n=5, Etrasimod n=5). *p<0.05, **p<0.01, ***p<0.001.
Figure 6
Figure 6
S1P1 and S1P2 are upregulated in palmitate-treated BMDM. (A) In BMDM, expression of S1P1 and S1P2 mRNA was induced following palmitate treatment whereas the abundance of S1P4 remained unchanged (vehicle n=4, palmitate n=4). (B) In PMH, S1P1, S1P2, S1P3 and S1P5 were detected and remained unchanged with palmitate treatment (vehicle n=3, palmitate n=3). (C) In whole livers, S1PR2 was upregulated, S1PR5 was downregulated, and no change was noted in S1PR1 and S1PR3 in mice fed the FFC diet in comparison to CD fed controls. CD (n=4) and FFC (n=5). *p<0.05, **p<0.01, ***p<0.001.
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