Phenotypical and biochemical characterization of murine psoriasiform and fibrotic skin disease models in Stabilin-deficient mice.

Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are scavenger receptors expressed by liver sinusoidal endothelial cells (LSECs). The Stabilin-mediated scavenging function is responsible for regulating the molecular composition of circulating blood in mammals. Stab1 and Stab2 have been shown to influence fibrosis in liver and kidneys and to modulate inflammation in atherosclerosis. In this context, circulating and localized TGFBi and POSTN are differentially controlled by the Stabilins as their receptors. To assess Stab1 and Stab2 functions in inflammatory and fibrotic skin disease, topical Imiquimod (IMQ) was used to induce psoriasis-like skin lesions in mice and Bleomycin (BLM) was applied subcutaneously to induce scleroderma-like effects in the skin. The topical treatment with IMQ, as expected, led to psoriasis-like changes in the skin of mice, including increased epidermal thickness and significant weight loss. Clinical severity was reduced in Stab2-deficient compared to Stab1-deficient mice. We did not observe differential effects in the skin of Stabilin-deficient mice after bleomycin injection. Interestingly, treatment with IMQ led to a significant increase of Stabilin ligand TGFBi plasma levels in Stab2-/- mice, treatment with BLM resulted in a significant decrease in TGFBi levels in Stab1-/- mice. Overall, Stab1 and Stab2 deficiency resulted in minor alterations of the disease phenotypes accompanied by alterations of circulating ligands in the blood in response to the disease models. Stabilin-mediated clearance of TGFBi was altered in these disease processes. Taken together our results suggest that Stabilin deficiency-associated plasma alterations may interfere with preclinical disease severity and treatment responses in patients.

Association of Differentially Altered Liver Fibrosis with Deposition of TGFBi in Stabilin-Deficient Mice.

Liver sinusoidal endothelial cells (LSECs) control clearance of Transforming growth factor, beta-induced, 68kDa (TGFBi) and Periostin (POSTN) through scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2). Stabilin inhibition can ameliorate atherosclerosis in mouse models, while Stabilin-double-knockout leads to glomerulofibrosis. Fibrotic organ damage may pose a limiting factor in future anti-Stabilin therapies. While Stab1-deficient (Stab1-/-) mice were shown to exhibit higher liver fibrosis levels upon challenges, fibrosis susceptibility has not been studied in Stab2-deficient (Stab2-/-) mice. Wildtype (WT), Stab1-/- and Stab2-/- mice were fed experimental diets, and local ligand abundance, hepatic fibrosis, and ligand plasma levels were measured. Hepatic fibrosis was increased in both Stab1-/- and Stab2-/- at baseline. A pro-fibrotic short Methionine-Choline-deficient (MCD) diet induced slightly increased liver fibrosis in Stab1-/- and Stab2-/- mice. A Choline-deficient L-amino acid-defined (CDAA) diet induced liver fibrosis of similar distribution and extent in all genotypes (WT, Stab1-/- and Stab2-/-). A hepatic abundance of Stabilin ligand TGFBi correlated very highly with liver fibrosis levels. In contrast, plasma levels of TGFBi were increased only in Stab2-/- mice after the CDAA diet but not the MCD diet, indicating the differential effects of these diets. Here we show that a single Stabilin deficiency of either Stab1 or Stab2 induces mildly increased collagen depositions under homeostatic conditions. Upon experimental dietary challenge, the local abundance of Stabilin ligand TGFBi was differentially altered in Stabilin-deficient mice, indicating differentially affected LSEC scavenger functions. Since anti-Stabilin-directed therapies are in clinical evaluation for the treatment of diseases, these findings bear relevance to treatment with novel anti-Stabilin agents.

Deficiency for scavenger receptors Stabilin-1 and Stabilin-2 leads to age-dependent renal and hepatic depositions of fasciclin domain proteins TGFBI and Periostin in mice.

Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are two major scavenger receptors of liver sinusoidal endothelial cells that mediate removal of diverse molecules from the plasma. Double-knockout mice (Stab-DKO) develop impaired kidney function and a decreased lifespan, while single Stabilin deficiency or therapeutic inhibition ameliorates atherosclerosis and Stab1-inhibition is subject of clinical trials in immuno-oncology. Although POSTN and TFGBI have recently been described as novel Stabilin ligands, the dynamics and functional implications of these ligands have not been comprehensively studied. Immunofluorescence, Western Blotting and Simple Western™ as well as in situ hybridization (RNAScope™) and qRT-PCR were used to analyze transcription levels and tissue distribution of POSTN and TGFBI in Stab-KO mice. Stab-POSTN-Triple deficient mice were generated to assess kidney and liver fibrosis and function in young and aged mice. TGFBI and POSTN protein accumulated in liver tissue in Stab-DKO mice and age-dependent in glomeruli of Stabilin-deficient mice despite unchanged transcriptional levels. Stab-POSTN-Triple KO mice showed glomerulofibrosis and a reduced lifespan comparable to Stab-DKO mice. However, alterations of the glomerular diameter and vascular density were partially normalized in Stab-POSTN-Triple KO. TGFBI and POSTN are Stabilin-ligands that are deposited in an age-dependent manner in the kidneys and liver due to insufficient scavenging in the liver. Functionally, POSTN might partially contribute to the observed renal phenotype in Stab-DKO mice. This study provides details on downstream effects how Stabilin dysfunction affects organ function on a molecular and functional level.

Targeting of Scavenger Receptors Stabilin-1 and Stabilin-2 Ameliorates Atherosclerosis by a Plasma Proteome Switch Mediating Monocyte/Macrophage Suppression.

BACKGROUND: Scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are preferentially expressed by liver sinusoidal endothelial cells. They mediate the clearance of circulating plasma molecules controlling distant organ homeostasis. Studies suggest that Stab1 and Stab2 may affect atherosclerosis. Although subsets of tissue macrophages also express Stab1, hematopoietic Stab1 deficiency does not modulate atherogenesis. Here, we comprehensively studied how targeting Stab1 and Stab2 affects atherosclerosis. METHODS: ApoE-KO mice were interbred with Stab1-KO and Stab2-KO mice and fed a Western diet. For antibody targeting, Ldlr-KO mice were also used. Unbiased plasma proteomics were performed and independently confirmed. Ligand binding studies comprised glutathione-S-transferase-pulldown and endocytosis assays. Plasma proteome effects on monocytes were studied by single-cell RNA sequencing in vivo, and by gene expression analyses of Stabilin ligand-stimulated and plasma-stimulated bone marrow-derived monocytes/macrophages in vitro. RESULTS: Spontaneous and Western diet-associated atherogenesis was significantly reduced in ApoE-Stab1-KO and ApoE-Stab2-KO mice. Similarly, inhibition of Stab1 or Stab2 by monoclonal antibodies significantly reduced Western diet-associated atherosclerosis in ApoE-KO and Ldlr-KO mice. Although neither plasma lipid levels nor circulating immune cell numbers were decisively altered, plasma proteomics revealed a switch in the plasma proteome, consisting of 231 dysregulated proteins comparing wildtype with Stab1/2-single and Stab1/2-double KO, and of 41 proteins comparing ApoE-, ApoE-Stab1-, and ApoE-Stab2-KO. Among this broad spectrum of common, but also disparate scavenger receptor ligand candidates, periostin, reelin, and TGFBi (transforming growth factor, ß-induced), known to modulate atherosclerosis, were independently confirmed as novel circulating ligands of Stab1/2. Single-cell RNA sequencing of circulating myeloid cells of ApoE-, ApoE-Stab1-, and ApoE-Stab2-KO mice showed transcriptomic alterations in patrolling (Ccr2-/Cx3cr1++/Ly6Clo) and inflammatory (Ccr2+/Cx3cr1+/Ly6Chi) monocytes, including downregulation of proatherogenic transcription factor Egr1. In wildtype bone marrow-derived monocytes/macrophages, ligand exposure alone did not alter Egr1 expression in vitro. However, exposure to plasma from ApoE-Stab1-KO and ApoE-Stab2-KO mice showed a reverted proatherogenic macrophage activation compared with ApoE-KO plasma, including downregulation of Egr1 in vitro. CONCLUSIONS: Inhibition of Stab1/Stab2 mediates an anti-inflammatory switch in the plasma proteome, including direct Stabilin ligands. The altered plasma proteome suppresses both patrolling and inflammatory monocytes and, thus, systemically protects against atherogenesis. Altogether, anti-Stab1- and anti-Stab2-targeted therapies provide a novel approach for the future treatment of atherosclerosis.