Lipopolysaccharide-induced Maturation of Bone Marrow-derived Dendritic Cells Is Regulated by Notch Signaling through the Up-regulation of CXCR4

Abstract

Dendritic cells (DCs) are professional antigen presenting cells to initiate immune response against pathogens, but mechanisms controlling the maturation of DCs are unclear. Here we report that, in the absence of recombination signal binding protein-Jκ (RBP-J, the transcription factor mediating Notch signaling), lipopolysaccharide-stimulated monocyte-derived DCs are arrested at a developmental stage with few dendrites, low major histocompatibility complex II (MHC II) expression, and reduced motility and antigen presentation ability. RBP-J null DCs had lower expression of CXCR4. Transduction with a CXCR4-expressing lentivirus rescued developmental arrest of RBP-J-deficient DCs. Activation of Notch signaling in DCs up-regulated CXCR4 expression and increased the outgrowth of dendrites and the expression of MHC II. These effects were abrogated by a CXCR4 inhibitor. Therefore, Notch signaling is essential for DCs to transit from a dendritelowMHC IIlow immature state into a dendritehighMHC IIhigh mature state, during the lipopolysaccharide-induced DC maturation, most likely through the up-regulation of CXCR4. Dendritic cells (DCs) are professional antigen presenting cells to initiate immune response against pathogens, but mechanisms controlling the maturation of DCs are unclear. Here we report that, in the absence of recombination signal binding protein-Jκ (RBP-J, the transcription factor mediating Notch signaling), lipopolysaccharide-stimulated monocyte-derived DCs are arrested at a developmental stage with few dendrites, low major histocompatibility complex II (MHC II) expression, and reduced motility and antigen presentation ability. RBP-J null DCs had lower expression of CXCR4. Transduction with a CXCR4-expressing lentivirus rescued developmental arrest of RBP-J-deficient DCs. Activation of Notch signaling in DCs up-regulated CXCR4 expression and increased the outgrowth of dendrites and the expression of MHC II. These effects were abrogated by a CXCR4 inhibitor. Therefore, Notch signaling is essential for DCs to transit from a dendritelowMHC IIlow immature state into a dendritehighMHC IIhigh mature state, during the lipopolysaccharide-induced DC maturation, most likely through the up-regulation of CXCR4. Dendritic cells (DCs) 4The abbreviations used are:DCsdendritic cellsRTreverse transcriptionMLRmixed lymphocyte reactionDTHdelayed-type hypersensitivitySEMscanning electron microscopeGSIγ-secretase inhibitorLPSlipopolysaccharideFACSfluorescence-activated cell sortingSSCside-scattering counterRBP-Jrecombination signal binding protein-JκBMbone marrowCFSEcarboxyfluorescein diacetate succinimidylesterNICNotch intracellular domainMHCmajor histocompatibility complexDllDelta-likeDSLDelta-Serrate-Lag2ILinterleukinGM-CSFgranulocyte macrophage-colony stimulating factorNF-κBnuclear factor κ-light-chain-enhancer of activated B cellsMEKmitogen-activated protein kinase/extracellular signal-regulated kinaseFITCfluorescein isothiocyanateGFPgreen fluorescent proteinEGFPenhanced GFPBrdUrdbromodeoxyuridineTrxthioredoxin. are professional antigen-presenting cells that initiate specific immune responses against pathogens such as bacteria, viruses, fungi, and parasites (1.Moll H. Cell. Microbiol. 2003; 5: 493-500Crossref PubMed Scopus (85) Google Scholar). Although several types of DCs participate in host resistance to microbial infections (2.Randolph G.J. Ochando J. Partida-S'anchez S. Annu. Rev. Immunol. 2008; 26: 293-316Crossref PubMed Scopus (381) Google Scholar), myeloid DCs play a primary role to reject microbes invading into tissues. The precursors of myeloid DCs move to the sites of infection using signaling through chemotactic receptors. Immature DCs, which are offspring of DC precursors, recognize microbial structures (pathogen-associated molecular patterns) using pattern recognition receptors (3.Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (6359) Google Scholar) such as Toll-like receptors, and capture antigens by phagocytosis, micropinocytosis, and endocytosis. Antigen uptake triggers the maturation of DCs, which is characterized by decreased endocytosis capacity, up-regulation of MHC II and co-stimulatory molecules, outgrowth of dendrites, and modulation of chemokine receptor expression patterns to facilitate the migration of antigen-loaded DCs to the T-cell zone in local lymph nodes (4.Ardavín C. Martínez del Hoyo G. Martín P. Anjuére F. Arias C.F. Marín A.R. Ruiz S. Parrillas V. Hernández H. Trends Immunol. 2001; 22: 691-700Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). These changes finally cause DCs to prime certain types of T-cell responses against the invading microbes. However, the mechanisms controlling DC maturation have not been fully understood. dendritic cells reverse transcription mixed lymphocyte reaction delayed-type hypersensitivity scanning electron microscope γ-secretase inhibitor lipopolysaccharide fluorescence-activated cell sorting side-scattering counter recombination signal binding protein-Jκ bone marrow carboxyfluorescein diacetate succinimidylester Notch intracellular domain major histocompatibility complex Delta-like Delta-Serrate-Lag2 interleukin granulocyte macrophage-colony stimulating factor nuclear factor κ-light-chain-enhancer of activated B cells mitogen-activated protein kinase/extracellular signal-regulated kinase fluorescein isothiocyanate green fluorescent protein enhanced GFP bromodeoxyuridine thioredoxin. The Notch signaling pathway is an evolutionarily highly conserved pathway that mediates direct cell-cell interaction and signaling (5.Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (5027) Google Scholar). Both Notch receptors and ligands are type I transmembrane proteins. When Notch receptors are triggered by ligands on neighboring cells, the intracellular domain of Notch receptors, or NIC, is released upon intra-membrane proteolysis executed by a γ-secretase-containing proteinase complex. NIC then translocates into the nuclear and associates with the DNA-binding protein recombination signal binding protein-Jκ (RBP-J) (6.Jarriault S. Brou C. Logeat F. Schroeter E.H. Kopan R. Israel A. Nature. 1995; 377: 355-358Crossref PubMed Scopus (1223) Google Scholar, 7.Tamura K. Taniguchi Y. Minoguchi S. Sakai T. Tun T. Furukawa T. Honjo T. Curr. Biol. 1995; 5: 1416-1423Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). This protein-protein interaction transactivates downstream genes associated with cell differentiation, proliferation, and apoptosis. Although four Notch receptors have been identified in mammals, it is believed that RBP-J mediates the transcriptional activation of all four Notch receptors (8.Honjo T. Genes Cells. 1996; 1: 1-9Crossref PubMed Scopus (183) Google Scholar). Notch signaling plays an important role in DC genesis. Cheng et al. (9.Cheng P. Nefedova Y. Miele L. Osborne B.A. Gabrilovich D. Blood. 2003; 102: 3980-3988Crossref PubMed Scopus (78) Google Scholar) showed that Notch1-deficient embryonic stem cells or hematopoietic progenitor cells were unable to differentiate into DCs. These authors further showed that different Notch ligands exhibited different effects on DC differentiation: Delta-like1 promoted the generation of fully differentiated DCs, whereas Jagged1 stimulated the accumulation of DC precursors but prevented their transition into terminally differentiated DCs (10.Cheng P. Nefedova Y. Corzo C.A. Gabrilovich D.I. Blood. 2007; 109: 507-515Crossref PubMed Scopus (74) Google Scholar). Other studies have shown roughly consistent findings showing that Notch signaling favors DC generation. For example, Weijzen et al. (11.Weijzen S. Velders M.P. Elmishad A.G. Bacon P.E. Panella J.R. Nickoloff B.J. Miele L. Kast W.M. J. Immunol. 2002; 169: 4273-4278Crossref PubMed Scopus (87) Google Scholar) reported that Jagged1 was able to induce the maturation of monocyte-derived human DCs, whereas Ohishi et al. showed that Delta-like1 inhibits the differentiation of monocytes into macrophages but permits their differentiation into DCs (12.Ohishi K. Varnum-Finney B. Flowers D. Anasetti C. Myerson D. Bernstein I.D. Blood. 2000; 95: 2847-2854Crossref PubMed Google Scholar, 13.Ohishi K. Varnum-Finney B. Serda R.E. Anasetti C. Bernstein I.D. Blood. 2001; 98: 1402-1407Crossref PubMed Scopus (104) Google Scholar). Recently, using a conditional knockout mouse of RBP-J and a DC-specific Cre transgenic mouse (CD11c-Cre), Caton et al. found that the deletion of RBP-J in DCs did not preclude DC lineage commitment, but could result in the reduction of spleen DC fraction, and specifically, could lead to the loss of spleen CD8− DCs (14.Caton M.L. Smith-Raska M.R. Reizis B. J. Exp. Med. 2007; 204: 1653-1664Crossref PubMed Scopus (651) Google Scholar). In addition, Notch signaling has also been implicated in the development of plasmacytoid DCs (15.Dontje W. Schotte R. Cupedo T. Nagasawa M. Scheeren F. Gimeno R. Spits H. Blom B. Blood. 2006; 107: 2446-2452Crossref PubMed Scopus (82) Google Scholar, 16.Olivier A. Lauret E. Gonin P. Galy A. Blood. 2006; 107: 2694-2701Crossref PubMed Scopus (53) Google Scholar). Mouse bone marrow (BM) cells can generate DCs when cultured in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4, mimicking the process of differentiation of monocytes into DCs (17.León B. Marténez del Hoyo G. Parrillas V. Vargas H.H. Sánchez-Mateos P. Longo N. López-Bravo M. Ardavín C. Blood. 2004; 103: 2668-2676Crossref PubMed Scopus (109) Google Scholar, 18.León B. Lopez-Bravo M. Ardavin C. Semin. Immunol. 2005; 17: 313-318Crossref PubMed Scopus (103) Google Scholar, 19.Fogg D.K. Sibon C. Miled C. Jung S. Aucouturier P. Littman D.R. Cumano A. Geissmann F. Science. 2006; 311: 83-87Crossref PubMed Scopus (800) Google Scholar, 20.Varol C. Landsman L. Fogg D.K. Greenshtein L. Gildor B. Margalit R. Kalchenko V. Geissmann F. Jung S. J. Exp. Med. 2007; 204: 171-180Crossref PubMed Scopus (493) Google Scholar). Winzler et al. have divided this differentiation process into three stages: immature stage (stage 1), mature stage (stage 2), and apoptotic stage (stage 3) (21.Winzler C. Rovere P. Rescigno M. Granucci F. Penna G. Adorini L. Zimmermann V.S. Davoust J. Ricciardi-Castagnoli P. J. Exp. Med. 1997; 185: 317-328Crossref PubMed Scopus (729) Google Scholar). In this study, using this system, we show that lipopolysaccharide (LPS)-induced maturation of monocyte-derived DCs is regulated by the Notch signaling through the up-regulation of CXCR4. Mice were maintained in a specific pathogen-free environment. The RBP-J-floxed mouse was described previously (22.Han H. Tanigaki K. Yamamoto N. Kuroda K. Yoshimoto M. Nakahata T. Ikuta K. Honjo T. Int. Immunol. 2002; 14: 637-645Crossref PubMed Scopus (491) Google Scholar). For the induction of RBP-J deletion, RBP-J-floxed mice and Mx-Cre mice were mated. One-month-old mice with suitable genotypes were induced with poly(I)-poly(C) as described (22.Han H. Tanigaki K. Yamamoto N. Kuroda K. Yoshimoto M. Nakahata T. Ikuta K. Honjo T. Int. Immunol. 2002; 14: 637-645Crossref PubMed Scopus (491) Google Scholar). Two months after the first injection, mice were used for further analysis. All animal experiments, including maintenance, injection, BM and were by and in with a from the of the to with the Mice were by cell were from cultured cells, or from mouse by through a by with were with for on and were using a were using the cells were by and cells were from was from The Delta-Serrate-Lag2 domain of was into the of which a by recombination of the as and the protein as a was the from and the of was as described F. H. 2008; PubMed Scopus Google Scholar). DCs were from BM cells using the or cultured DCs were in the and was to the was from the using a from was using a and the system, with as a used in were as and cell were by cells with the and were by and were were using CXCR4 or at by with or were using an enhanced on were in at were then and were in on with and a scanning electron microscope DCs K. M. N. H. M. S. S. J. Exp. Med. PubMed Scopus Google Scholar), mice were by BM was from and and was by through an and with a by in were and cultured in in of and and with mouse and mouse for the was the of the In experiments, the inhibitor or a CXCR4 inhibitor was at of or DCs were with carboxyfluorescein diacetate succinimidylester at for by with of for on were with and were into the of cells were from lymph nodes and then by were in cells with or P. and H. were in the lower DCs were on the in the and were cultured at in the lower were and and by was as described Google Scholar) by an in of on the of The lymph cells were and by The of the mouse CXCR4 was by from a mouse embryonic using and The CXCR4 was with an and was into by to the to generate and were into cells using with the to the were after the and the were by at at for were in and at For BM cells were with the for and then cultured in further analysis. DCs which were stimulated with were in with were mixed with DCs, and were to the of was by was by The was after and was used as E. Mice were in the with DCs with the E. was by Mice were and lymph nodes were were using and were by Mice were at with E. DCs. after the DC injection, the mice were by of of E. into the of four was using a The of the responses was by the The of the mouse CXCR4 was by from mouse The was into to generate a of the as in were also by and with the of CXCR4 to generate different and cells were cultured in with and were with of different of that NIC, and of using after the was using and a to the All was to inhibitor kinase inhibitor inhibitor inhibitor and inhibitor were all from was with the were as were using was the role of Notch signaling in DC maturation, we RBP-J-floxed mice Mx-Cre with mice as (22.Han H. Tanigaki K. Yamamoto N. Kuroda K. Yoshimoto M. Nakahata T. Ikuta K. Honjo T. Int. Immunol. 2002; 14: 637-645Crossref PubMed Scopus (491) Google Scholar). cultured BM cells from the RBP-J knockout and in the presence of and and stimulated DC maturation with K. M. N. H. M. S. S. J. Exp. Med. PubMed Scopus Google Scholar). BM cells DCs after cultured for in cell and during the were found the RBP-J knockout and cells not When and DCs were stimulated with and were for and we found that DCs were in a of cells with in shown in and on the of the and BM cells of DCs with low the and of the induced an of DCs from with a of DCs. In did not induce the of DCs from DCs decreased as in BM cell which with the outgrowth of dendrites of DCs. then outgrowth of DCs stimulated with DCs with dendrites and DCs with dendrites were an as shown in induced the of DCs with dendrites increased the of DCs with dendrites This of outgrowth with the in increased DCs with and decreased DCs with low that a transition of DCs into DCs the of DCs with DCs an shown in a DC had a with However, most DCs their dendrites, as by dendrites an and of the of dendrites of and DCs on different to a These that RBP-J essential for the maturation of DCs in of upon In to the expression of MHC II is of DC the expression of MHC II and DCs. DCs had lower MHC II expression DCs that DCs could divided into with low MHC II and the with MHC II. In DCs, the MHC was that the low expression of MHC II on DCs was to the of MHC IIhigh we MHC II expression DCs with low and with and in in the The result showed that on of the DCs in had a of MHC II expression with DCs in When DCs in and were and their MHC II expression was and DCs, we to in MHC II expression and DCs in or and Therefore, the deletion of RBP-J DC maturation at a state with low dendrites and low MHC II. However, DC molecules, such as and did not upon RBP-J deletion not DC maturation is by expression of chemokine receptors. In DCs, whereas the expression of was not not the expression CXCR4 was reduced upon when with the the that knockout of RBP-J CXCR4 expression in DCs. This was further by of cell of and DCs with which showed that DCs had lower of CXCR4 further the expression of CXCR4 on DCs from showed that, in BM and the of CXCR4 expression was reduced in DCs When DCs were using DC we found that cells from mice also showed reduced expression of as with the not the of CXCR4 and DCs using Both showed that DCs from mice had lower of CXCR4 the These to a that, in RBP-J knockout the expression of CXCR4 was reduced in DCs. CXCR4 expression a reduced chemotactic migration then the migration of and DCs in response to the of CXCR4. In a system, in cultured and DCs were in the and cells with an were cultured in the lower DCs in the lower were after the of the The result that DCs had lower in response to also in migration of and DCs by DCs into the of and DCs at the lymph nodes by shown in DCs at the lymph with DCs. we a which the migration of mature DCs in The result showed that a of DCs were with at the of not but DCs to the lymph nodes the of the RBP-J-deficient DCs using several shown in in mixed lymphocyte reaction cultured with DCs, which were activated with showed proliferation, with the the in antigen presentation capacity, mice were with or DCs, and the of in the lymph nodes was N. J. Exp. Med. PubMed Scopus Google Scholar, E. A. A. J. Exp. Med. 1997; 185: PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar). expression of CXCR4 for the maturation arrest of DCs, we the cultured DCs from BM of and mice with a lentivirus the mouse CXCR4 and with GFP as a cells and DCs from mice showed the expression of GFP and the mouse CXCR4 When cells were and we found that, the GFP expression did not the differentiation of or DCs, the of CXCR4-expressing rescued the outgrowth in DCs, as by DCs with the expression of CXCR4 also rescued MHC II expression in DCs Therefore, RBP-J maturation arrest of DCs could to the of CXCR4 RBP-J mediates Notch a Notch which could Notch signaling in a F. H. 2008; PubMed Scopus Google and not the expression of CXCR4 on DCs cultured in the presence of with cultured with the showed that DCs cultured with had a of CXCR4 on their DCs in the which Notch abrogated CXCR4 up-regulation in DCs, that the up-regulation of CXCR4 was on Notch we further showed that of Notch receptors could the expression of CXCR4 in DCs. in a mouse DC up-regulated the expression of CXCR4 on the cell and at the as shown by and then of Notch signaling DC maturation through outgrowth and MHC II When BM cells from mice were cultured in the presence of and and stimulated with of could the of DCs also found that, in the presence of DCs dendrites DCs cultured with the This was by outgrowth of DCs by a of cell with and showed that, the of DCs with did not show the of DCs with in was DCs in the after These that Notch receptors increased DCs with of MHC II expression showed that of Notch receptors with increased the expression of MHC II on the of DCs we BM cells with cells in the presence of and with as a 2002; 17: Full Text Full Text PDF PubMed Scopus Google Scholar). DC maturation was stimulated with as shown in and with and of DCs with the The expression of CXCR4 and MHC II on DCs cultured on were also with the These further that Notch promoted DC the of an inhibitor of CXCR4 on DC maturation in the presence of Notch DCs were cultured from BM cells of mice in the presence of which promoted DC differentiation, as shown in and described CXCR4 inhibitor the outgrowth and as as the up-regulation of MHC II induced by These further that the induction of DC maturation by Notch activation could on CXCR4 Notch signaling could the CXCR4 we the of the of the mouse CXCR4 by The was into the to generate or cells with and and the of the mouse CXCR4 by NIC using shown in in all three types of cells, of the induced the expression of from in a that NIC could the CXCR4 further the that to Notch we the CXCR4 that or to Notch signaling were in the These that, the the CXCR4 could or by Notch studies have shown that Notch signaling is not essential for lineage of DCs (14.Caton M.L. Smith-Raska M.R. Reizis B. J. Exp. Med. 2007; 204: 1653-1664Crossref PubMed Scopus (651) Google Scholar). we found that, when BM cells from RBP-J knockout mice were cultured in in the presence of and a of DCs could at a in of DC further characterized changes of RBP-J null DCs and found that RBP-J-deficient DCs dendrites, lower of MHC II molecules, and reduced of migration and antigen presentation in and in the cultured DCs with activated Notch signaling could the outgrowth and MHC II expression, that Notch promoted DC the changes during DC differentiation, we found that the of Notch signaling arrested DC maturation at a stage the outgrowth of dendrites and the up-regulation of MHC II molecules, of Notch signaling promoted DC differentiation by through this in and and DCs were not that Notch signaling essential for the DC of DCs is to their differentiation and the differentiation from from to DCs. 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PubMed Scopus Google Scholar). cells of Notch including it is likely that monocytes Notch as their through are to this have to the molecular mechanisms the Notch differentiation transition of DCs. a of showing that the chemokine receptor CXCR4 is downstream to Notch or and is for Notch differentiation transition of DCs. from and using in cultured DCs, DCs, as as a DC have shown that the expression of CXCR4 is in RBP-J-deficient DCs and up-regulated in a when DCs were stimulated with Notch using we found that the expression of CXCR4 rescued RBP-J differentiation of DCs. Notch DC differentiation is by a specific inhibitor of CXCR4. These a signaling from Notch receptor to chemokine receptor which is for DC have to the of up-regulation of CXCR4. Although that Notch signaling the CXCR4 this is most likely to In the of which is by NIC, is RBP-J binding The Notch up-regulation of CXCR4 not on P. A. V. S. Osborne B. Gabrilovich D.I. J. Immunol. 2001; PubMed Scopus (183) Google Scholar), the of by a specific inhibitor did not up-regulation of CXCR4 in DCs by Notch signaling not CXCR4 expression through or of did not abrogated Notch CXCR4 up-regulation the that Notch signaling and CXCR4 signaling have in the development of immune system, system, as as stem cells, it is not that mechanisms in the of CXCR4 by Notch Recently, et al. M. L. G. 2008; PubMed Scopus Google Scholar) reported that in human cells and cells, of the human Notch in the of human CXCR4 expression and the of a human CXCR4 is to the and as as in the transcriptional of the CXCR4 the professional antigen-presenting cells, DCs are in of human including infections and DC maturation is highly in cell immature DCs are reported in this have shown that Notch signaling is essential for the maturation of at of DCs, and Notch receptors can outgrowth and MHC II expression on DCs. Both outgrowth and MHC II expression are essential for DC antigen antigen findings have important in DC maturation in and in DCs in cell J. C. for and and for lentivirus with