Biol. Chem. 2015; 396(6-7): 775–782
Christina Dillmanna, Javier Moraa, Catherine Olesch, Bernhard Brüne and Andreas Weigert*
S1PR4 is required for plasmacytoid dendritic cell differentiation Abstract: The sphingolipid sphingosine-1-phosphate (S1P) has various functions in immune cell biology, regulating survival, proliferation, and, most prominently, migration. S1P couples to five G protein-coupled receptors (S1PR1–5) to transduce its effects on immune cell function. Expression of S1PR4 is restricted to immune cells. However, its impact on immune cell biology is largely elusive. In the current study, we intended to answer the question of whether S1P might affect plasmacytoid dendritic cell (pDC) migration, which dominantly express S1PR4. pDC are highly specialized cells producing large amounts of type I interferon in response to TLR7/9 ligands after viral infection or during autoimmunity. Surprisingly, we noticed a reduced abundance of pDC, particularly CD4- pDC, in all organs of S1PR4deficient vs. wildtype mice. This effect was not caused by altered migration of mature pDC, but rather a reduced potential of pDC progenitors, especially common DC progenitors, to differentiate into pDCs. In vitro studies suggested that reduced S1PR4-deficient pDC progenitor differentiation into mature pDC might be explained by both migration and differentiation of pDC progenitors in the bone marrow. As S1PR4 also affected the differentiation of CD34+ human hematopoietic stem cells into pDC, interfering with S1PR4 might be useful to reduce pDC numbers during autoimmunity. Keywords: common DC progenitor; hematopoiesis; migration; sphingosine-1-phosphate; stem cells.
a These authors contributed equally to this work. *Corresponding author: Andreas Weigert, Institute of Biochemistry I – Pathobiochemistry, Faculty of Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany, e-mail: [email protected] Christina Dillmann, Catherine Olesch and Bernhard Brüne: Institute of Biochemistry I – Pathobiochemistry, Faculty of Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany Javier Mora: Institute of Biochemistry I – Pathobiochemistry, Faculty of Medicine, Goethe University Frankfurt, Theodor-SternKai 7, D-60590 Frankfurt, Germany; and Faculty of Microbiology, University of Costa Rica, Costa Rica
DOI 10.1515/hsz-2014-0271 Received November 11, 2014; accepted January 9, 2015; previously published online January 13, 2015
Introduction Plasmacytoid dendritic cells (pDC) constitute a specialized dendritic cell (DC) population that produces high amounts of type I interferon in response to TLR7/9 activation. pDC differentiate in the bone marrow (BM) from both lymphoid and myeloid progenitors (Shortman et al., 2013). The lymphoid branch of pDC development is currently ill-defined. However, a decrease of BM pDC numbers in IL-7R-α null mice suggests common lymphoid progenitors (CLP) as one possible origin for pDC (Vogt et al., 2009) and cell culture experiments suggest that CLP differentiate to pDC via a progenitor with B cell potential (Sathe et al., 2013). The myeloid branch of pDC generation starts with common myeloid progenitors (CMP), which give rise to common monocyte/dendritic cell progenitors (MDP) followed by differentiation to either monocytes or common dendritic cell progenitors (CDP) (Shortman et al., 2013). The generation of CDP is fms-like tyrosine kinase 3 (Flt3)dependent, as mice lacking Flt3 show lower DC numbers compared to wildtype mice (McKenna et al., 2000). CDP finally differentiate into either pDC or pre-conventional DCs (pre-cDC), which migrate into the circulation to populate distinct organs. Sphingosine-1-phosphate (S1P) is a lipid mediator triggering lymphocyte cell trafficking by signaling through its specific G protein-coupled receptors (S1PR1–5) (Cyster and Schwab, 2012). S1P-dependent migration has been demonstrated for cDC (Rathinasamy et al., 2010). However its involvement in pDC migration remains unclear. Murine blood pDC express exclusively S1PR4 (Gao et al., 2009). S1PR4 expression is restricted to lymphoid and hematopoietic tissue and S1PR4-deficient (S1PR4-/-) mice show defects in megakaryocyte differentiation and DC function (Golfier et al., 2010; Schulze et al., 2011). We questioned whether pDC trafficking and function was altered in S1PR4-/- mice. Surprisingly, we observed reduced pDC numbers in S1PR4-/- mice, which was at least in part connected to S1PR4-dependent migration of pDC progenitors.
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776 C. Dillmann et al.: S1PR4 and pDC differentiation
Results Reduced pDC numbers in S1PR4-/- mice We were initially interested in whether S1P might affect plasmacytoid DC migration. Of all five S1P receptors, blood pDC express mainly S1PR4 (Gao et al., 2009). We validated this pattern in BM pDC that were isolated by magnetic bead sorting combined with FACS sorting using SiglecH as a specific pDC marker (Blasius et al., 2006). In the BM, pDC expressed mainly S1PR4, together with low levels of S1PR1, 2 and 5, whereas S1PR3 was not expressed (Supplementary Figure S2). Therefore, we aimed to compare migration of wildtype (WT) vs. S1PR4-/- pDC towards S1P. However, when isolating pDC from S1PR4-/- BM, we noticed a marked reduction in pDC numbers compared to wildtype controls (Figure 1A,B). pDCs can be divided into several immature as well as mature subpopulations. CCR9 expression is found on mature pDC, whereas SiglecH+ CCR9- cells are pDC precursors that retain remarkable cDC potential (Schlitzer et al., 2012; Shortman et al., 2013). Other markers such as expression of CD2 or CD4 determine functional mature subpopulations (O’Keeffe et al., 2002; Matsui et al.,
2009). When analyzing pDC subpopulations by polychromatic FACS in detail, we noticed that selectively the CD4pDC subpopulation of mature pDC was reduced in BM of S1PR4-/- mice compared to WT mice (Figure 1B,C). Importantly, other major immune cell populations in the BM were not significantly altered (Figure 1D). CD4- pDC are the immediate precursors of CD4+ pDC (O’Keeffe et al., 2002) and were indicated as the main migratory pDC subset in vivo (Yang et al., 2005). As CD4- CCR9+ migratory pDC numbers were specifically reduced in the BM of S1PR4-/mice, we hypothesized that the lack of S1PR4 expression would result in enhanced emigration of these cells into the circulation and subsequently into peripheral tissues. This would indicate S1PR4 as a negative regulator of pDC migration. However, when we investigated pDC numbers in peripheral tissues such as spleen (Figure 2A–C), liver, lymph nodes, and thymus (data not shown), as well as the blood (Figure 2E–G), we noticed a phenotype corresponding to the BM with reduced total pDC numbers, especially lower CD4- pDC numbers in S1PR4-/- compared to WT animals. Other major immune cell populations were not affected by S1PR4-deficiency (Figure 2D,H). These data indicated a developmental impact of S1PR4 on pDC rather than an impact on migration.
Figure 1 Reduced pDC numbers in S1PR4-/- BM. Total BM from two femurs of wildtype (WT) and S1PR4-/- (KO) mice was analyzed by polychromatic flow cytometry (six animals in each group, analyzed in three independent experiments). (A) The number of total pDC in BM is displayed. (B) Representative FACS plots indicating the relative abundance of SiglecH+ pDCs and specific subsets. (C) The number of mature (CCR9+) pDC subsets in BM is displayed. (D) The number of major BM immune cell subsets. (A,C,D) Data are mean±SEM. Data were analyzed by Student’s t-test (A) or two-way ANOVA with Bonferroni’s correction (C,D). *p
Christina Dillmanna, Javier Moraa, Catherine Olesch, Bernhard Brüne and Andreas Weigert*
S1PR4 is required for plasmacytoid dendritic cell differentiation Abstract: The sphingolipid sphingosine-1-phosphate (S1P) has various functions in immune cell biology, regulating survival, proliferation, and, most prominently, migration. S1P couples to five G protein-coupled receptors (S1PR1–5) to transduce its effects on immune cell function. Expression of S1PR4 is restricted to immune cells. However, its impact on immune cell biology is largely elusive. In the current study, we intended to answer the question of whether S1P might affect plasmacytoid dendritic cell (pDC) migration, which dominantly express S1PR4. pDC are highly specialized cells producing large amounts of type I interferon in response to TLR7/9 ligands after viral infection or during autoimmunity. Surprisingly, we noticed a reduced abundance of pDC, particularly CD4- pDC, in all organs of S1PR4deficient vs. wildtype mice. This effect was not caused by altered migration of mature pDC, but rather a reduced potential of pDC progenitors, especially common DC progenitors, to differentiate into pDCs. In vitro studies suggested that reduced S1PR4-deficient pDC progenitor differentiation into mature pDC might be explained by both migration and differentiation of pDC progenitors in the bone marrow. As S1PR4 also affected the differentiation of CD34+ human hematopoietic stem cells into pDC, interfering with S1PR4 might be useful to reduce pDC numbers during autoimmunity. Keywords: common DC progenitor; hematopoiesis; migration; sphingosine-1-phosphate; stem cells.
a These authors contributed equally to this work. *Corresponding author: Andreas Weigert, Institute of Biochemistry I – Pathobiochemistry, Faculty of Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany, e-mail: [email protected] Christina Dillmann, Catherine Olesch and Bernhard Brüne: Institute of Biochemistry I – Pathobiochemistry, Faculty of Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany Javier Mora: Institute of Biochemistry I – Pathobiochemistry, Faculty of Medicine, Goethe University Frankfurt, Theodor-SternKai 7, D-60590 Frankfurt, Germany; and Faculty of Microbiology, University of Costa Rica, Costa Rica
DOI 10.1515/hsz-2014-0271 Received November 11, 2014; accepted January 9, 2015; previously published online January 13, 2015
Introduction Plasmacytoid dendritic cells (pDC) constitute a specialized dendritic cell (DC) population that produces high amounts of type I interferon in response to TLR7/9 activation. pDC differentiate in the bone marrow (BM) from both lymphoid and myeloid progenitors (Shortman et al., 2013). The lymphoid branch of pDC development is currently ill-defined. However, a decrease of BM pDC numbers in IL-7R-α null mice suggests common lymphoid progenitors (CLP) as one possible origin for pDC (Vogt et al., 2009) and cell culture experiments suggest that CLP differentiate to pDC via a progenitor with B cell potential (Sathe et al., 2013). The myeloid branch of pDC generation starts with common myeloid progenitors (CMP), which give rise to common monocyte/dendritic cell progenitors (MDP) followed by differentiation to either monocytes or common dendritic cell progenitors (CDP) (Shortman et al., 2013). The generation of CDP is fms-like tyrosine kinase 3 (Flt3)dependent, as mice lacking Flt3 show lower DC numbers compared to wildtype mice (McKenna et al., 2000). CDP finally differentiate into either pDC or pre-conventional DCs (pre-cDC), which migrate into the circulation to populate distinct organs. Sphingosine-1-phosphate (S1P) is a lipid mediator triggering lymphocyte cell trafficking by signaling through its specific G protein-coupled receptors (S1PR1–5) (Cyster and Schwab, 2012). S1P-dependent migration has been demonstrated for cDC (Rathinasamy et al., 2010). However its involvement in pDC migration remains unclear. Murine blood pDC express exclusively S1PR4 (Gao et al., 2009). S1PR4 expression is restricted to lymphoid and hematopoietic tissue and S1PR4-deficient (S1PR4-/-) mice show defects in megakaryocyte differentiation and DC function (Golfier et al., 2010; Schulze et al., 2011). We questioned whether pDC trafficking and function was altered in S1PR4-/- mice. Surprisingly, we observed reduced pDC numbers in S1PR4-/- mice, which was at least in part connected to S1PR4-dependent migration of pDC progenitors.
Brought to you by | Oakland University Authenticated Download Date | 6/3/15 11:40 PM
776 C. Dillmann et al.: S1PR4 and pDC differentiation
Results Reduced pDC numbers in S1PR4-/- mice We were initially interested in whether S1P might affect plasmacytoid DC migration. Of all five S1P receptors, blood pDC express mainly S1PR4 (Gao et al., 2009). We validated this pattern in BM pDC that were isolated by magnetic bead sorting combined with FACS sorting using SiglecH as a specific pDC marker (Blasius et al., 2006). In the BM, pDC expressed mainly S1PR4, together with low levels of S1PR1, 2 and 5, whereas S1PR3 was not expressed (Supplementary Figure S2). Therefore, we aimed to compare migration of wildtype (WT) vs. S1PR4-/- pDC towards S1P. However, when isolating pDC from S1PR4-/- BM, we noticed a marked reduction in pDC numbers compared to wildtype controls (Figure 1A,B). pDCs can be divided into several immature as well as mature subpopulations. CCR9 expression is found on mature pDC, whereas SiglecH+ CCR9- cells are pDC precursors that retain remarkable cDC potential (Schlitzer et al., 2012; Shortman et al., 2013). Other markers such as expression of CD2 or CD4 determine functional mature subpopulations (O’Keeffe et al., 2002; Matsui et al.,
2009). When analyzing pDC subpopulations by polychromatic FACS in detail, we noticed that selectively the CD4pDC subpopulation of mature pDC was reduced in BM of S1PR4-/- mice compared to WT mice (Figure 1B,C). Importantly, other major immune cell populations in the BM were not significantly altered (Figure 1D). CD4- pDC are the immediate precursors of CD4+ pDC (O’Keeffe et al., 2002) and were indicated as the main migratory pDC subset in vivo (Yang et al., 2005). As CD4- CCR9+ migratory pDC numbers were specifically reduced in the BM of S1PR4-/mice, we hypothesized that the lack of S1PR4 expression would result in enhanced emigration of these cells into the circulation and subsequently into peripheral tissues. This would indicate S1PR4 as a negative regulator of pDC migration. However, when we investigated pDC numbers in peripheral tissues such as spleen (Figure 2A–C), liver, lymph nodes, and thymus (data not shown), as well as the blood (Figure 2E–G), we noticed a phenotype corresponding to the BM with reduced total pDC numbers, especially lower CD4- pDC numbers in S1PR4-/- compared to WT animals. Other major immune cell populations were not affected by S1PR4-deficiency (Figure 2D,H). These data indicated a developmental impact of S1PR4 on pDC rather than an impact on migration.
Figure 1 Reduced pDC numbers in S1PR4-/- BM. Total BM from two femurs of wildtype (WT) and S1PR4-/- (KO) mice was analyzed by polychromatic flow cytometry (six animals in each group, analyzed in three independent experiments). (A) The number of total pDC in BM is displayed. (B) Representative FACS plots indicating the relative abundance of SiglecH+ pDCs and specific subsets. (C) The number of mature (CCR9+) pDC subsets in BM is displayed. (D) The number of major BM immune cell subsets. (A,C,D) Data are mean±SEM. Data were analyzed by Student’s t-test (A) or two-way ANOVA with Bonferroni’s correction (C,D). *p
