Role of the co-stimulatory molecule inducible T-cell co-stimulator ligand (ICOSL) in the progression of experimental metabolic dysfunction-associated steatohepatitis

Abstract

Background and aims: Inducible T-cell Co-Stimulator (ICOS) present on T-lymphocytes and its ligand ICOSL expressed by myeloid cells play multiple roles in regulating T-cell functions. However, recent evidence indicates that reverse signalling involving ICOSL is also important in directing the differentiation of monocyte-derived cells. In this study, we investigated the involvement of ICOS/ICOSL dyad in modulating macrophage functions during the evolution of metabolic dysfunction-associated steatohepatitis (MASH).

Results: In animal models of MASH, ICOS was selectively up-regulated on CD8⁺ T-cells in parallel with an expansion of ICOSL-expressing macrophages. An increase in circulating soluble ICOSL was also evident in patients with MASH as compared to healthy individuals. ICOSL knockout (ICOSL^-/-) mice receiving choline/methionine deficient (MCD) diet for 6 weeks had milder steatohepatitis than wild type mice. MASH improvement was confirmed in mice fed with cholesterol-enriched Western diet for 24 weeks in which ICOSL deficiency greatly reduced liver fibrosis along with the formation of crown-like macrophage aggregates producing the pro-fibrogenic mediators osteopontin (OPN) and galectin-3 (Gal-3). These effects associated with a selective shewing of F4-80⁺/CD11b^high monocyte-derived macrophages (MoMFs) expressing the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) to CD11b^low/F4-80⁺ cells positive for the Kupffer cell marker C-type lectin-like type 2 receptor (CLEC-2), thus indicating an increased MoMF maturation toward monocyte-derived Kupffer cells.

Conclusions: These results suggest that CD8⁺ T-cells interaction with monocyte-derived macrophages through ICOS/ICOSL critically supports a specific subset of TREM2⁺-expressing cells contributing to the evolution of steatohepatitis. The data also point ICOS/ICOSL dyad as a possible target for therapeutic interventions in MASH.

Keywords: chronic inflammation; galectin-3; liver fibrosis; macrophages; metabolic dysfunction-associated steatotic liver disease; nonalcoholic fatty liver disease; osteopontin.

Figure 1

MASH associates with changes in the liver distribution of ICOS and ICOSL expressing cells. Wild type C57BL/6 mice received a methionine/choline deficient (MCD) or control diets for 6 weeks and hepatic lymphocytes and macrophages were analyzed by flow cytometry. (A) ICOS expression by the whole pool of CD3⁺ T lymphocytes and localization of ICOS-positive T cell in relation to the distribution of CD4⁺ helper and CD8⁺ cytotoxic and double negative T-cells. (B) ICOSL up-regulation within liver myeloid cells and localization of ICOSL-positive cells among CD11b⁺/F4-80 hepatic macrophages. The values refer to 4-5 animals per group and the boxes include the values within 25^th and 75^th percentile, while the horizontal bars represent the medians. The extremities of the vertical bars (10^th-90^th percentile) comprise 80% percent of the values.

Figure 2

An elevation of the soluble form of ICOSL characterize human MASLD/MASH. (A) The soluble forms ICOSL (sICOSL) were measured by ELISA assays in the sera of 81 (48 males and 33 females) patients with and 40 age/gender matched healthy subjects. (B) Distribution of sICOSL values according to the severity of liver disease as evaluated by the NAFLD activity score (NAS) and by the grading of hepatic fibrosis among MASLD/MASH patients.

Figure 3

ICOSL ablation improves liver injury and inflammation in mice with MASH. Wild type (WT) and ICOSL deficient (ICOSL ko) mice were fed with either control or methionine choline deficient (MCD) diets for 6 weeks and the severity of steatohepatitis was investigated by: (A) Haematoxylin/eosin staining of liver sections (Magnification 200x); (B) Alanine aminotransferase (ALT) release; The hepatic mRNA levels of the leucocyte marker CD11b (C) and of inflammatory markers TNF-α, CXCL10, IL-12p40 and TREM-1 (D). RT-PCR values are expressed as fold increase of 2^-ΔCT over the relative control samples. The values in the panels B, and C refer to 5-7 animals per group and the boxes include the values within 25^th and 75^th percentile, while the horizontal bars represent the median. The extremities of the vertical bars (10^th-90^th percentile) include 80% of the values.

Figure 4

ICOSL modulates progression to liver fibrosis of experimental MASH. Wild type (WT) and ICOSL deficient (ICOSL ko) mice were fed with either control or Western diet (WD) diets for 24 weeks and the severity of steatohepatitis was investigated by: (A) Haematoxylin/eosin (HE) staining of liver sections (Magnification 200x); (B) Alanine aminotransferase (ALT) release; (C) liver triglyceride content; (D) hepatic mRNA levels of inflammatory markers CD11b and IL-12p40. The extent of hepatic fibrosis was evaluated by measuring (E) fibrosis markers α1-procollagen, α-smooth muscle actin (α-SMA) and Transforming Growth Factor-β1 (TGF-β1) and (F) liver collagen deposition as detected by Sirius Red staining (SR). The transcripts for RT-PCR values are expressed as fold increase of 2^-ΔCT over the relative control samples. The values in the panels B-F refer to 5-8 animals per group and the boxes include the values within 25^th and 75^th percentile, while the horizontal bars represent the median. The extremities of the vertical bars (10^th-90^th percentile) include 80% of the values.

Figure 5

ICOSL deficiency reduced monocyte-derived macrophage distribution and crown-like macrophage aggregates in rodent MASH livers. Wild type (WT) and ICOSL deficient (ICOSL ko) mice were fed with either control or Western (WD) diets for 24 weeks and the animals were investigated for the distribution of hepatic macrophages. (A) Flow cytometry analysis of the distribution of Ly6C^high/CD11b^high/F4-80⁺ pro-inflammatory monocyte/macrophages (MoMFs) and CD206⁺/CD11b^low/F4-80⁺ macrophages (MPhs). (B) Immunofluorescence (IF) staining of macrophage crown-like structures (hCLSs) using anti-F4-80 antibodies (arrows). The values refer to 4 animals per group ± SD.

Figure 6

ICOSL signaling modulates macrophage phenotype in rodent MASH livers. Wild type (WT) and ICOSL deficient (ICOSL ko) mice were fed with either control or Western (WD) diets for 24 weeks and investigated for NAM/SAM markers. (A) hepatic mRNA levels of TREM2, Galectin-3 (Gal-3) and Osteopontin (OPN). RT-PCR values are expressed as fold increase of 2^-ΔCT over the relative control samples. The values in the panel refer to 5-8 animals per group and the boxes include the values within 25^th and 75^th percentile, while the horizontal bars represent the median. The extremities of the vertical bars (10^th-90^th percentile) include 80% of the values. (B) Immunohistochemical detection of Gal-3 and OPN (arrows) in liver sections (magnification 20x). Quantification was performed by counting, respectively, Gal-3 positive hepatic crown-like structures (hCLS) or OPN positive cells in 10 microscopic fields at 20x magnification.

Figure 7

Interference with ICOS/ICOSL dyad selectively affects the distribution of TREM2 expressing cells among MASH-associated hepatic macrophages. (A) Distribution of TREM2⁺/CD9⁺ cells among CD11b^high/F4-80⁺ monocyte/macrophages (MoMFs) and CD11b^low/F4-80⁺ macrophages (Mphs) in the liver of wild type (WT) and ICOSL deficient (ICOSL ko) fed with either control or Western (WD) diets for 24 weeks. (B) Distribution of TREM2⁺/ICOSL⁺ cells among CD11b^high/F4-80⁺ monocyte/macrophages (MoMFs) and CD11b^low/F4-80⁺ macrophages (MPhs) in the liver of wild type (WT) and ICOS deficient (ICOS ko) fed with either control or Western (WD) diets for 24 weeks. The values refer to 4 animals per group ± SD.

Figure 8

ICOSL modulates monocyte maturation to monocyte derived Kupffer cells (moKCs). Wild type (WT) and ICOSL deficient (ICOSL ko) mice were fed with either control or methionine choline deficient (MCD) for 2 weeks and the animals were investigated by flow cytometry for the distribution of Kupffer cells markers CLEC2 and TIM4 (A) and the prevalence of TREM2^high cells (B) among CD11b^low/F4-80⁺ macrophages. The values refer to 4 animals per group ± SD.

Figure 9

Proposed mechanisms for the modulation of liver macrophages phenotype by ICOS/ICOSL interaction. ICOS expressing CD8⁺ T-cells can contribute to support pro-inflammatory and pro-fibrogenic TREM2⁺/ICOSL⁺ MoMFs involved in forming crown-like aggregates by preventing their differentiation to monocyte-derived Kupffer cells. Image created with BioRender.com.