S1PR1 on tumor-associated macrophages promotes lymphangiogenesis and metastasis via NLRP3/IL-1ß.

Metastasis is the primary cause of cancer death. The inflammatory tumor microenvironment contributes to metastasis, for instance, by recruiting blood and lymph vessels. Among tumor-infiltrating immune cells, tumor-associated macrophages (TAMs) take a center stage in promoting both tumor angiogenesis and metastatic spread. We found that genetic deletion of the S1P receptor 1 (S1pr1) alone in CD11bhi CD206+ TAMs infiltrating mouse breast tumors prevents pulmonary metastasis and tumor lymphangiogenesis. Reduced lymphangiogenesis was also observed in the nonrelated methylcholanthrene-induced fibrosarcoma model. Transcriptome analysis of isolated TAMs from both entities revealed reduced expression of the inflammasome component Nlrp3 in S1PR1-deficient TAMs. Macrophage-dependent lymphangiogenesis in vitro was triggered upon inflammasome activation and required both S1PR1 signaling and IL-1ß production. Finally, NLRP3 expression in tumor-infiltrating macrophages correlated with survival, lymph node invasion, and metastasis of mammary carcinoma patients. Conceptually, our study indicates an unappreciated role of the NLRP3 inflammasome in promoting metastasis via the lymphatics downstream of S1PR1 signaling in macrophages.

PGE2/EP4 signaling in peripheral immune cells promotes development of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory autoimmune disease model of multiple sclerosis (MS). The inflammatory process is initiated by activation and proliferation of T cells and monocytes and by their subsequent migration into the central nervous system (CNS), where they induce demyelination and neurodegeneration. Prostaglandin E2 (PGE2) - synthesized by cyclooxygenase 2 (COX-2) - has both pro- and anti-inflammatory potential, which is translated via four different EP receptors. We hypothesized that PGE2 synthesized in the preclinical phase by peripheral immune cells exerts pro-inflammatory properties in the EAE model. To investigate this, we used a bone marrow transplantation model, which enables PGE2 synthesis or EP receptor expression to be blocked specifically in peripheral murine immune cells. Our results reveal that deletion of COX-2 or its EP4 receptor in bone marrow-derived cells leads to a significant delay in the onset of EAE. This effect is due to an impaired preclinical inflammatory process indicated by a reduced level of the T cell activating interleukin-6 (IL-6), reduced numbers of T cells and of the T cell secreted interleukin-17 (IL-17) in the blood of mice lacking COX-2 or EP4 in peripheral immune cells. Moreover, mice lacking COX-2 or EP4 in bone marrow-derived cells show a reduced expression of matrix metalloproteinase 9 (MMP9), which results in decreased infiltration of monocytes and T cells into the CNS. In conclusion, our data demonstrate that PGE2 synthesized by monocytes in the early preclinical phase promotes the development of EAE in an EP4 receptor dependent manner.

Prostacyclin mediates neuropathic pain through interleukin 1ß-expressing resident macrophages.

Prostacyclin is an important mediator of peripheral pain sensation. Here, we investigated its potential participation in mediating neuropathic pain and found that prostacyclin receptor (IP) knockout mice exhibited markedly decreased pain behavior. Application of an IP antagonist to the injury site or selective IP deficiency in myeloid cells mimicked the antinociceptive effect observed in IP knockout mice. At the site of nerve injury, IP was expressed in interleukin (IL) 1ß-containing resident macrophages, which were less common in IP knockout mice. Local administration of the IP agonist cicaprost inhibited macrophage migration in vitro and promoted accumulation of IP- and IL1ß-expressing cells as well as an increase of IL1ß concentrations at the application site in vivo. Fittingly, the IL1-receptor antagonist anakinra (IL-1ra) decreased neuropathic pain behavior in wild-type mice but not in IP knockout mice. Finally, continuous, but not single administration, of the cyclooxygenase inhibitor meloxicam early after nerve injury decreased pain behavior and the number of resident macrophages. Thus, early synthesis of prostacyclin at the site of injury causes accumulation of IL1ß-expressing macrophages as a key step in neuropathic pain after traumatic injury.

Apoptotic cells enhance sphingosine-1-phosphate receptor 1 dependent macrophage migration.

The lipid sphingosine-1-phosphate (S1P) is a chemokine for a variety of immune cells including lymphocytes and monocytes. Migration toward S1P is determined by the S1P receptor expression profile, with S1PR1/3 (where S1PR is S1P receptor) stimulating and S1PR2 attenuating migration. However, the impact and physiological significance of S1P-induced migration of macrophages is largely unclear. We observed that alternative activation of human macrophages, by IL-4 or apoptotic cells (ACs), enhanced S1PR1 expression. Moreover, ACs provoked macrophage migration toward S1P in an S1PR1-dependent manner as confirmed by pharmacological receptor inhibition and S1PR1-deficient murine macrophages. In a mouse model of resolving peritoneal inflammation, F4/80-driven deletion of S1PR1 reduced postinflammatory macrophage emigration from inflammatory sites. S1PR1 expression on macrophages might, therefore, be relevant for restoring tissue homeostasis during the resolution of inflammation.

HIF-1α is a negative regulator of plasmacytoid DC development in vitro and in vivo.

Hypoxia-inducible factors (HIFs) regulate hematopoiesis in the embryo and maintain hematopoietic stem cell function in the adult. How hypoxia and HIFs contribute to hematopoietic lineage differentiation in the adult is ill defined. Here we provide evidence that HIF-1 limits differentiation of precursors into plasmacytoid dendritic cells (pDCs). Low oxygen up-regulated inhibitor of DNA binding 2 (ID2) and suppressed Flt3-L-induced differentiation of bone marrow cells to pDCs in wild-type but not HIF-1α(fl/fl) LysM-Cre bone marrow cells. Moreover, pDC differentiated normally in hypoxic ID2(-/-) bone marrow cultures. Finally, we observed elevated pDC frequencies in bone marrow, blood, and spleen of HIF-1α(fl/fl) LysM-Cre and ID2(-/-), but not HIF-2α(fl/fl) LysM-Cre mice. Our data indicate that the low oxygen content in the bone marrow might limit pDC development. This might be an environmental mechanism to restrict the numbers of these potentially autoreactive cells.

S1P regulation of macrophage functions in the context of cancer.

The sphingolipid sphingosine-1-phosphate (S1P) is an important regulator of immune cell functions in vivo. Besides recruiting lymphocytes to blood and lymph, it may promote immune cell survival and proliferation, but also interferes with their activation. Hereby, S1P may act as an intracellular second messenger or cofactor or, upon being secreted from cells, may bind to and activate a family of specific G-protein-coupled receptors (S1PR1-5). Extracellular versus intracellular S1P hereby might trigger synergistic/identical or fundamentally distinct responses. Furthermore, engagement of different S1PRs is connected to different functional outcome. This complexity is exemplified by the influence of S1P on the inflammatory potential of macrophages, shaping their role in inflammatory pathologies such as atherosclerosis and cancer. Here, we summarize the recent progress in understanding the impact of S1P signaling in macrophage biology, discuss its impact in solid as well as 'wet' tumors and elaborate potential options to interfere with S1P signaling in the context of cancer.