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Retinol Saturase Knock-Out Mice Are Characterized by Impaired Clearance of Apoptotic Cells and Develop Mild Autoimmunity

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Retinol Saturase Knock-Out Mice Are Characterized by Impaired Clearance of Apoptotic Cells and Develop Mild Autoimmunity

Zsolt Sarang et al. Biomolecules.

Abstract

Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvβ35 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.

Keywords: MFG-E8; autoimmunity; efferocytosis; macrophage; neuropeptide Y; retinol saturase.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyzes, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Loss of retinol saturase (RetSat) does not affect the expression of retinoid-regulated efferocytosis-related genes or the thymic apopto-phagocytosis program in mice. (A) 4 week old mice were injected intraperitoneally with 0.3 mg dexamethasone acetate (DEX) or vehicle to induce thymocyte apoptosis. After 24 h, thymi were collected and gene expression was determined by qRT-PCR (n = 4) using β-actin as a normalizing gene. WT, wild type; KO, knock-out. Results are expressed as mean ± SD (n = 4). (B) The number of various thymocyte cell populations in the thymi of 4 week old wild type and RetSat-null mice. Results are expressed as mean ± SD (n = 4). (C) Percentage of annexin-V-positive thymocytes 6 h after apoptosis induction by 1 μM DEX in vitro. (D,E) The number of various thymocyte cell populations in the thymi of 4 week old wild type and RetSat-null mice at 24 h after exposure to 0.3 mg DEX (D) or 5 Gy irradiation (E). Results are expressed as mean ± SD (n = 4).
Figure 2
Figure 2
Loss of RetSat delays the long-term efferocytosis of both bone-marrow-derived macrophages (BMDMs) and peritoneal macrophages. 5(6)-carboxyfluorescein diacetate (CFDA-SE)-labelled (A) BMDMs or (B) peritoneal macrophages were exposed to Deep-Red-dye-labelled apoptotic thymocytes for 1 h (in a 1:5 macrophage—target-cell ratio) either immediately (short-term phagocytosis) or after 5 h of engulfing non-labelled apoptotic thymocytes. The number of ingested cells was determined by confocal microscopy. Upper panels show one representative experiment. Total number of ingested cell results in lower panels are expressed as mean ± SD (n = 3), * p < 0.05.
Figure 3
Figure 3
Loss of RetSat leads to decreased MFG-E8 mRNA expression in various macrophages. (A) Expression of MFG-E8 in wild type and RetSat-null BMDMs, peritoneal macrophages, and thioglycolate-elicited macrophages as compared to wild type BMDMs. These macrophages were generated as described in Materials and Methods. (B) Relative MFG-E8 mRNA expression of wild type and RetSat-null BMDMs before and after 5 h efferocytosis as compared to the non-engulfing wild type BMDMs. (C) Normalized mRNA expressions of RetSat, MFG-E8, TG2, and NPY during differentiation of wild type and RetSat-null monocytes in the presence and absence of 1 μM all-trans-13,14- dihydroretinol (DR-OH). (D) Induction of retinoid-sensitive efferocytosis genes at the end of the 5 day monocyte differentiation in wild type and RetSat-null cells in the presence of 1 μM all-trans-13,14- dihydroretinol, as compared to the vehicle-treated control cells. mRNA expressions in all these experiments were determined by qRT-PCR using β-actin as a normalizing gene. Results are expressed as mean ± SD (n = 4), * p < 0.05. (E) Short-term phagocytosis of wild type and RetSat-null BMDMs differentiated in the presence or absence of 1 μM all-trans-13,14-dihydroretinol. Percentage of engulfing macrophages was determined by flow cytometry analysis. Results are expressed as mean ± SD (n = 4), * p < 0.05. One representative confocal image is also shown. CFDA-SE-labelled macrophages appear as green cells, while Deep Red dye-labelled apoptotic thymocytes appear as red cells. Engulfed apoptotic thymocytes appear with orange color, as the green and red colors overlap.
Figure 4
Figure 4
Impaired efferocytosis of RetSat-null macrophages is related to improper MFG-E8 production. Long-term efferocytosis of wild type and RetSat-null (A) BMDMs and (B) peritoneal macrophages was determined in the presence and absence of 2 μg/mL recombinant MFG-E8 protein. CFDA-SE-labelled macrophages were exposed to Deep Red dye-labelled apoptotic thymocytes for 1 h after continuous engulfing of non-labelled apoptotic thymocytes for 5 h. The number of ingested cells was determined by confocal microscopy. Upper panels show one representative experiment; total ingested cell number results in lower panels are expressed as mean ± SD (n = 3). (C) 4 week old mice were injected daily i.p. daily with 1 mg/kg DEX for 6 days to induce MFG-E8 expression and then exposed to 0.5 Gy irradiation to trigger thymocyte apoptosis. After 8 h, thymi were collected and the number of various thymocyte cell populations was determined by FACS analysis. Results are expressed as mean ± SD (n = 4), * p < 0.05.
Figure 5
Figure 5
Aged female RetSat-null mice develop mild autoimmunity. (A) Splenic weights of 1 year old female wild type and RetSat-null mice. * p < 0.05. (B) Immunohistochemical detection of activated caspase-3-positive cells in the spleens of 6 wild type and the 6 RetSat-null mice with enlarged spleens was carried out as described in Materials and Methods. Data represent mean ± SD of the number of activated caspase-3-positive cells/high power field, * p < 0.05. (C) Levels of anti-dsDNA antibodies in the serum of wild type and RetSat-null mice are expressed as mean ± SD. OD; optical density. (D) Anti-nuclear antibodies (ANA) in the serum of 12 wild type and 12 RetSat-null mice. Images demonstrate the fluorescence intensity for the ANA scores from 0 to 4. (E) Immune complex deposits (shown with green color) in the kidneys of aged female RetSat-null mice. Cell nuclei are stained blue with 4′,6-diamidino-2-phenylindole (DAPI).

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