Hematopoietic sphingosine 1-phosphate lyase deficiency decreases atherosclerotic lesion development in LDL-receptor deficient mice

Abstract

Aims: Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/-)) deficiency on leukocyte subsets relevant to atherosclerosis.

Methods and results: LDL receptor deficient mice that were transplanted with Sgpl1(-/-) bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1(-/-) chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response.

Conclusions: Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.

Figure 1. Detailed outline of the experimental setup.

Figure 2. Recipient bone marrow genotyping and S1P concentrations.

Genotyping of the bone marrow (A) showed >90% repopulation of the bone marrow for Sgpl1. (B) Effect of hematopoietic Sgpl1^−/− on plasma S1P concentration and S1P content of thymus, lymph node, and spleen. White bars: wild-type (WT), black bars: Sgpl1^−/− (KO) chimeras. Data are represented as mean +/− SEM^−/−, data from n = 10–11 per group in total. *P<0.05, **P<0.01, ***P<0.001 (WT versus KO).

Figure 3. Effect of hematopoietic Sgpl1^−/− on T cell numbers.

(A,B) Sgpl1^−/− chimeras show reduced CD4⁺ and CD8⁺ T cell numbers in blood, spleen and lymph nodes. (C) CD3 staining shows absence of normal germinal centre morphology in spleens of Sgpl1^−/− chimeras (scale bar: 100 µm, arrows indicate germinal centers in spleens of WT transplanted mice). (D). Sgpl1^−/− chimeras show a relative increase in regulatory T cells in spleen and lymph nodes. White bars: wild-type (WT), black bars: Sgpl1^−/− (KO) chimeras. Data are represented as mean ± SEM, n = 10–11. *P<0.05, **P<0.01, ***P<0.001 (WT versus KO).

Figure 4. Effect of hematopoietic S1P lyase deficiency on T-cell proliferation and migration.

(A) The mitotic response of T cells isolated from Sgpl1^−/− chimeras to αCD3/αCD28 (2 µg/mL) or ConA (2 µg/mL) challenge is markedly decreased compared to Sgpl1^+/+ transplanted mice (n = 8 per group). (B) In vivo proliferation of CD4⁺ T cells as measured by EdU incorporation is also significantly decreased in the Sgpl1^−/− chimeras (n = 8 per group). (C, D) CCR7, CXCR4 and S1P₁ positive cells were overrepresented in spleen CD4⁺ and CD8⁺ T-cell subsets of Sgpl1^−/− (n = 10–11 per group). (E) Splenocytes of CCL19-challenged Sgpl1^+/+ transplanted mice but not Sgpl^−/− chimeras show ConA induced IL-2 release, which corresponds to reduced proliferation (n = 4 per group) (F). (G) Flow cytometry analysis of lymphoid organs and circulation at 48 hours after injection of a 1∶1 mixture of CMTMR labeled Sgpl1^−/− versus CFSE labeled wild type lymphocytes into LDLr^−/− revealed a reduced presence of Sgpl1^−/− lymphocytes in spleen, lymph nodes and blood (WBC, white blood cells) (n = 9). This reduced homing was confirmed by fluorescence microscopic analysis of lymph node (H) and spleen (I), (scale bar: 100 µm, Sgpl1^−/− lymphocytes in red, wild type lymphocytes in green). White bars: wild-type (WT), black bars: Sgpl1^−/− (KO) chimeras. Data are represented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (WT versus KO), ^###P<0.001 (PBS versus CCL19).

Figure 5. Altered myelopoiesis in Sgpl1^−/−chimeras.

(A) Sgpl1^−/−chimeras show increased CD11b⁺ (monocytes) and CD11b⁺Gr1⁺CD71⁻ neutrophil counts in the circulation (n = 10–11 per group). (B) In bone marrow the increase in neutrophils is already noticeable at the level of the CD11b⁺/Gr1⁺ precursors, while this is not the case for the CD14⁺/GR1⁺ monocytes (n = 10–11 per group). (C) In vivo proliferation of CD11b⁺ cells as measured by EdU incorporation is 3-fold increased in Sgpl1^−/− chimeras (n = 8 per group). (D) Growth-stimulation of bone marrow cells indicated an increased colonigenic capacity of Sgpl1^−/− cells in response to G-CSF and GM-CSF, while no effect for M-CSF was seen (n = 3 per group). (E) Bone marrow-derived macrophages of Sgpl1^−/−chimeras are unresponsive to S1P stimulus as assessed by Ca²⁺-flux measurement, while the Ca²⁺ response to ionomycin and MCP-1 remained unaltered (n = 3 per group). (F). Analysis of plasma and bone marrow-derived macrophage (BM-mφ) supernatants pointed to reduced MCP-1 generation in Sgpl1^−/−chimeras (plasma: n = 10–11 per group, BM-mφ: n = 5 per group). Reduced MCP1 expression by BM derived macrophages of Sgpl1^−/− chimeras was corroborated at an mRNA levels (G), while conversely expression of CCR2 was increased (H) in bone marrow-derived macrophages (n = 10 per group). However, in a model of mild peritonitis, MCP-1 triggered migration of CCR2⁺ inflammatory macrophages (F4/80, P<0.05) in Sgpl1^+/+ transplanted mice but not Sgpl1^−/− chimeras (n = 4–5 per group) (I). White bars: wild-type (WT), black bars: Sgpl1^−/− (KO) chimeras. Data are represented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (WT versus KO).

Figure 6. Characterization of macrophage phenotype in Sgpl1^+/+ transplanted mice versus Sgpl1^−/− chimeras.

Gene expression analysis of bone marrow derived macrophages indicated a generally pro-inflammatory phenotype of Sgpl1^−/− macrophages as judged by the increased expression of the markers IL-6 (A), TNF-α (B), IL-1α (C) and IL-12 (D), while in contrast iNOS expression, another marker of a pro-inflammatory “classically activated” macrophage was sharply decreased (E). Two markers of the anti-inflammatory macrophage phenotype, notably arginase 1 (Arg1) (F) and IL-10 (G) were almost blunted in LPS primed macrophages. Expression of the established M2 marker IL-1RA (H) was increased in control macrophages, while LPS stimulation caused a shift towards downregulated expression in Sgpl1^−/− macrophages. (I) In addition, LPS-induced TNF-α secretion was increased in BM-mφ of Sgpl1^−/− chimera mice. (J) Furthermore, LPS slightly induced the release of the pro-inflammatory IL-12 in control macrophages. The endogenous levels of IL-12 were already higher in Sgpl1^−/− macrophages; however in contrast to control macrophages, LPS did not enhance IL-12 production of these macrophages. White bars: wild-type (WT), black bars: Sgpl1^−/− (KO) chimeras. Data are represented as mean ± SEM, gene expression: n = 10 per group, cytokine analysis: n = 5 per group. *P<0.05, **P<0.01, ***P<0.001 (WT versus KO).

Figure 7. Effect of Sgpl1^−/− chimerism on aortic root lesion size and morphology.

(A) Lesion analysis of aortic root lesions shows that absence of hematopoietic S1P lyase decreases atherosclerotic lesion progression compared to control animals by 35% (scale bar: 250 µm). (B) A trend was observed towards a decreased T-cell content in the aortic root (P = 0.09) (scale bar: 10 µm). (C) Despite the observed systemic monocytosis, immunohistochemical analysis did not demonstrate any changes in macrophage content as demonstrated by a MOMA-2 staining and the general macrophage marker CD163. A trend was observed towards decreased plaque expression of the M1 macrophage marker iNOS in Sgpl1^−/− chimeras (P = 0.055) (scale bar: 250 µm, nuclear DAPI staining in blue, macrophage marker staining in red). (D) Macrophage polarization markers such as CCR2 (freshly invaded monocytes) and TIMP3 and MMP14 (foam cells) were unchanged (scale bar: 250 µm, nuclear DAPI staining in blue, macrophage marker staining in red). White bars: wild-type (WT), black bars: Sgpl1^−/− (KO) chimeras, n = 10–11 per group. Data are represented as mean ± SEM. *P<0.05 (WT versus KO).