Intrinsic retinoic acid signaling determines the

development of macrophages and dendritic

cells in the marginal zone of the spleen

Marieke R. Beijer1, Aymeric Rivollier2, Georg Kraal1, Reina E. Mebius1, William W. Agace2, Joke M.M. den Haan1

1 Department of Molecular Cell Biology and Immunology, VU University Medical Center, P.O. Box 7057, Amsterdam, The Netherlands

2 Immunology Section, Lund University, Lund 221 84, Sweden

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AbstractVitamin A is essential for the generation of efficient mucosal immune responses against harmful pathogens. In addition, vitamin A also influences the development of various immune cells that are located in the marginal zone of the spleen. We previously showed that the development of marginal zone B cells and Notch2 dependent CD4+ and DN dendritic cells (DCs) is dependent on vitamin A. In contrast, the development of marginal zone macrophages (MZMs) is inversely correlated with the levels of vitamin A and retinoic acid in the diet. To determine whether intrinsic vitamin A signaling determines DC and macrophage development, we investigated the development of DCs and MZMs in mice that overexpress dominant negative retinoic acid receptor alpha (RARα) in CD11c+ cells leading to deficient vitamin A signaling. Vitamin A signaling was necessary for the development of ESAM+ Notch2 dependent CD4+ and DN DCs. Furthermore, SIGNR1+ MZM were expanded when vitamin A signaling was abrogated. Taken together, our study shows that the development of CD4+ and DN DCs, and MZMs in the spleen is regulated by intrinsic vitamin A signaling and show previously unknown effects of vitamin A in the development and function of the immune system.

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IntroductionDendritic cells (DCs) are essential for activation of the adaptive immune response. In steady state, murine splenic DCs can be divided into plasmacytoid (pDCs) and conventional DCs (cDCs), which are further divided into three distinct subsets. CD4+ and double negative (DN (CD4-CD8-)) DCs are located in the bridging channels and marginal zone of the spleen and are characterized by the expression of CD11b and Sirpα. They are specialized in the presentation of antigens by major histocompatibility complex II (MHC-II) to CD4+ T cells. In contrast, CD8+ DCs are located in the red pulp and the T cell zones of the spleen and specifically express DEC205 and CD24. CD8+ DCs are specialized in cross-presentation of cell associated and viral antigens to CD8+ T cells 1,2. Previous studies indicated that various cell types are involved in the development of DCs and macrophages in the spleen. For example, B cells provide a lymphotoxin-α1β2 signal that stimulates the development of CD4+ and DN DCs 3. Furthermore, Notch signaling is required for the development of CD4+ and DN DCs and MZ B cells in the spleen. Reticular fibroblasts that are located in the red pulp area of the spleen express Notch ligands Dll1, Dll4, Jagged1 and Jagged2. Also F4/80+ red pulp macrophages express Dll1, Jagged1 and Jagged2 4,5.

Besides DCs, several macrophage populations are located in the marginal zone of the spleen. CD169+ macrophages (also referred to as marginal metallophilic macrophages or sialoadhesin+ macrophages) are positioned adjacent to the MAdCAM1+ rim of sinus lining cells that marks the marginal zone 6. CD169+ macrophages bind pathogens and immune complexes, transfer these to B cells, and thereby stimulate humoral immune responses 7-9. In addition, CD169+ macrophages produce type I interferons and other inflammatory cytokines upon bacterial and viral infection, activate innate effector cells and prevent further dissemination of the infection 10-16. A ring of marginal zone macrophages (MZMs) is located closer to the red pulp and surrounds the CD169+ macrophages. All MZMs express scavenger receptor MARCO (macrophage receptor with collagenous structure), while the transmembrane protein SIGNR1 (specific ICAM3 grabbing nonintegrin-related 1) is only expressed by a subset of MZMs 17,18. The development of MZMs is dependent on the growth factor macrophage colony stimulating factor (M-CSF) and the presence of MZ  B  cells 18,19. MZMs are essential for the clearance of polysaccharide encapsulated bacteria such as Streptococcus pneumoniae and Neisseria meningitides 20-22. Furthermore, MZMs are crucial for the elimination of apoptotic cells and regulation of tolerance 23-25. Therefore, MZMs are essential to activate immune responses against various pathogens and for maintaining immune homeostasis.

Vitamin A (retinol) has long been recognized for its role in immunity. The most recent estimates from the world health organization, estimate that approximately 190 million preschool children and 20 million pregnant mothers are vitamin A deficient 26. This affects immunity, sight and embryonic development. Vitamin A is taken up from the diet and stored in the liver, which regulates systemic vitamin A levels. When necessary, the liver releases vitamin A bound to retinol binding proteins (RBP) in the circulation and the complex is taken up by cells expressing receptors for RBP. In the cell vitamin A is

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converted in two subsequent oxidation steps into its biologically active form retinoic acid (RA) by the enzymes retinol dehydrogenase (ADH) and retinal dehydrogenase (RALDH). The RA that is produced can have autocrine or paracrine activity by binding to retinoic acid receptor and retinoic X receptor (RAR-RXR) heterodimers that are bound to retinoic acid response elements (RAREs) present in the promoter regions of various genes 27.

Emerging evidence shows that vitamin A is important for functioning of the mucosal immune system, but also the development of immune cells such as marginal zone B cells and DC subsets in the spleen is vitamin A dependent 28,29. We recently showed that the development of splenic Notch2 dependent CD4+ and DN DCs is dependent on vitamin A 28. Furthermore, we observed an inverse correlation between dietary vitamin A levels and the development of MARCO+SIGNR1+ macrophages in the spleen (chapter 3). However, the mechanisms that underlie this were not completely elucidated. In these studies, dietary vitamin A levels were manipulated which resulted in different levels of vitamin A and its active metabolite RA in the blood and in changes in immune cell populations. Since MZ B cells are virtually absent in spleens of VAD mice, the reduction in CD4+ and DN DCs in these mice could be a secondary effect caused by the reduction in MZ B cells. On the other hand, CD4+ and DN DCs express RALDH2 that converts vitamin A metabolite retinal into its biologically active form: retinoic acid (RA). Furthermore, RARα,β,γ and RXRα,β are expressed at higher levels in CD4+ and DN DCs than CD8+ DCs 28. Thus, it remains to be determined whether vitamin A directly affects DC and macrophage development or whether the differentiation of these cell types are indirectly regulated.

To determine the effect of RA on CD11c+ DCs and macrophages, RA signaling was selectively inhibited in CD11c+ cells. A dominant negative form of human retinoic acid receptor alpha (RARα) (RAR403) (hereafter denoted as dnRARα) lacking the AF2 domain that is required for ligand dependent activation has previously been generated and used to study the effect of inhibition of RA signaling 30. dnRARα, was overexpressed in CD11c+ cells by breeding dnRARαflox/flox mice with CD11c-Cre mice. The resulting CD11c-CreposdnRARα mice that express dnRARα in CD11c+ cells, were compared to CD11c-CrenegdnRARα mice that did not express dnRARα. We observed that intrinsic vitamin A signaling is crucial for the development of CD4+ and DN DCs. Furthermore, in mice overexpressing dnRARα, the development of MARCO+SIGNR1+ macrophages, that also express CD11c, was increased. In contrast, the development of marginal zone B cells was not affected, which are also dependent on vitamin A, but do not express CD11c. Together, these data indicate that intrinsic vitamin A signaling is essential for the development of CD4+ and DN DCs and MZMs in the spleen.

Materials and methodsMiceMice overexpressing a dominant negative form of RARα retinoid receptor (RAR403) were previously reported 30. Mice expressing a construct containing a neomycin resistance gene and three polyadenylation sequences flanked by loxP sites upspstream of the RAR403

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locus targeted in the ROSA26 locus were crossed with CD11c-Cre+/- mice. The resulting Cre+ mice overexpressed dnRARα in all CD11c expressing cells (hereafter denoted as CD11c-CreposdnRARα). CD11c-CrenegdnRARα littermates that did not express CD11c-Cre recombinase were used as negative control. The ethical committee of Lund University approved all animal experiments.

DC isolation and flow cytometrySpleens were isolated and digested with liberase TL (Roche Diagnostics GmbH, Mannheim, Germany) as previously described 31. Cells were incubated with 2.4G2 supernatant to block aspecific binding of the antibodies. Cells were stained in PBS supplemented with 0.5% BSA. The following antibodies were purchased from eBioscience (San Diego, CA): CD11c (clone N418), Sirpα (CD172α, clone P84), CD3ε (clone 145-2C11), CD19 (clone eBio1D3), Ter119 (clone TER-119), NK1.1 (clone PK136), B220 (clone RA3-6B2), Rat IgG2a (clone eBR2a). CD4 (clone GK1.5), CD8 (clone 53-6.7), CD135 (clone a2F10) were obtained from BD Bioscience (Breda, The Netherlands). ESAM (clone 1G8/ESAM) was purchased at Biolegend. Secondary antibody was streptavidin-PerCP purchased at BD Bioscience (Breda, The Netherlands). Cells were analyzed with a FACScanto (Becton Dickinson, Franklin Lakes, NJ). Data analysis was performed using FlowJo 9.2 (Tree Star).

Immunofluorescence histologySpleens were embedded in O.C.T. TM medium (Tissue-Tek, Sakura) and frozen at -80°C until further use. Cryosections (5 μm) were collected on gelatine coated glass slides and were fixed in dehydrated acetone for 2 minutes and air-dried. Sections were blocked with 5% mouse serum in PBS prior to antibody staining. Sections were embedded in a mixture of polyvinylalcohol and glycerol supplemented with DAPI (Invitrogen) and analyzed on a Leica DM6000 fluorescence microscope Microscope (Leica Microsystems, Netherland b.v., Rijswijk, The Neterlands).

RNA isolation, cDNA synthesis and real time PCRTotal RNA was isolated from total spleen homogenates using TRIzol reagent (Invitrogen Life Technologies, Breda, The Netherlands). RNA was isolated by precipitation with isopropanol and cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Fermentas Life Sciences, Vilnius, Lithuania) according to manufacturers’ protocol. Real time PCR was performed using SYBR Green Mastermix (PE Applied Biosystems, Foster City, CA) on an ABI Prism 7900HT Sequence Detection System (PE Applied Biosystems Foster City, CA). A standard curve was generated using pooled lymph node tissue to correct for primer efficiency. mRNA quantities were normalized to HPRT. The following primers were used: HPRT Fw: 5’-CCTAAGATGAGCGCAAGTTGAA-3’,  HPRT Rev: 5’-CCACAGGACTAGAACACCTGCTAA-3’, MARCO Fw: 5’-GGCCTTCCAGGAAACAAAGG-3’, MARCO Rev: 5’-CTCCTGGAGATCCAGCCAAA-3’, SIGNR1 Fw: 5’-CTGAACTCTTGTCCAGG ATTCCC-3’, SIGNR1 Rev: 5’-GGCAGAGTCGACACAGGCGGA-3’.

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Statistical analysisStatistical significance was tested using GraphPad Prism 4 (La Jolla, CA) by performing a two-tailed Student’s T-test or ANOVA with Bonferroni’s correction. A P value of 0.05 was considered to be significant.

ResultsIntrinsic retinoic acid signaling promotes the development of CD4+ DCs in the spleenPreviously, we showed that dietary vitamin A levels control the development of Notch2 dependent CD4+ and DN DCs in the spleen. In these studies, dietary vitamin A deficiency and retinoic acid (RA) supplementation were used to assess the effect of vitamin A. However, since these models alter vitamin A or RA levels in the systemic circulation, it was not possible to determine whether direct vitamin A signaling in splenic DCs and macrophages was responsible for this effect, or whether this was indirectly caused by effects on other cell populations. Therefore, our aim was to determine whether intrinsic vitamin A signaling by CD11c+ DCs is required for the development of CD4+ and DN DCs. For this, mice overexpressing a dominant negative mutant of human RARα in CD11c+ cells (CD11c-CreposdnRARα) were compared to littermates that did not express dnRARα (CD11c-CrenegdnRARα). Overexpression of dnRARα was confirmed by quantitative PCR (data not shown).

Figure 1. RA directly promotes the development of CD4+ DCs in the spleen. (A) Total numbers of Lin-(CD19-, NK1.1-, CD3-)MHC-II+CD11chigh DCs were determined in spleens of CD11c-CrenegdnRARα (white bar) and CD11c-CreposdnRARα (black bar) mice. DC numbers are given as the percentage of total live non-autofluorescent cell population. (B) pre-cDCs defined as Lin-(CD19-, NK1.1-, Ter119-, CD3-)MHC-II-CD11c+SirpαintCD135+ were analyzed in CD11c-CrenegdnRARα (white bar) and CD11c‑CreposdnRARα (black bar) mice. Total numbers of pre‑cDCs were depicted as percentage of live non‑autofluorescent cells. (C) Representative dot plots showing expression of CD4 and CD8 in Lin-MHC-II+CD11chigh DCs and the percentage of CD4+, CD8+ and DN DCs of total live non-autofluorescent cells. Data are shown as mean + SEM of 6 mice and are from one experiment representative of 2 performed. *P<0.05, **P<0.01, ***<0.001, ****<0.0001 ANOVA with Bonferroni’s correction or student’s T-test.

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Comparable to vitamin A deficient mice, a significant reduction of splenic cDCs (Lin-MHC-II+CD11chigh) was observed in mice overexpressing dnRARα in CD11c+ cells (figure 1A). cDCs develop from a common precursor that is present in the bone marrow and seeds the spleen and a significant reduction in pre-cDCs (Lin-CD11c+MHC-II-SirpαintCD135+) was observed in the spleen of CD11c-CreposdnRARα mice (figure 1B) indicating that intrinsic vitamin A signaling is required for maintaining a stable pre-cDC population in the spleen. This is in contrast to what was observed in vitamin A deficient mice in which the pre-cDC population was not significantly affected 28.

Subsequently, we determined the numbers of CD4+, CD8+ and DN DCs. We previously showed that CD4+ and DN DCs require vitamin A for their development, while CD8+ DCs develop in a vitamin A independent process 28. In mice where vitamin A signaling was impaired (CD11c-CreposdnRARα mice), a reduction of CD4+ DCs was observed. No differences in DN or CD8+ DC numbers were detected in mice overexpressing dnRARα (figure 1C). This indicates that while the development of CD4+ DCs requires intrinsic vitamin A signaling, DN DC numbers unexpectedly are not affected by abrogation of intrinsic vitamin A signaling.

Intrinsic vitamin A signaling promotes the development of Notch2 dependent DN and CD4+ DCsNotch signaling is essential for the development of CD4+ and DN DCs 32 and Notch2 dependency coincides with the expression of endothelial cell selective adhesion molecule (ESAM) 33. Recently, we showed that all CD4+ DCs and a subset of the DN DCs in the spleen express high levels of ESAM. Furthermore, we revealed that dietary vitamin A specifically stimulates the development of Notch2 dependent ESAM expressing CD4+ and DN DCs in the spleen 28. To determine whether vitamin A signaling in CD11c+ cells is required for the development of these Notch2 dependent DCs, we assessed ESAM expression on DCs of mice with impaired vitamin A signaling (CD11c-CreposdnRARα). Similar to what was observed in vitamin A deficient mice, while exclusively ESAMhighCD4+ DCs were observed in control mice, a substantial population of ESAMlowCD4+ DCs was detected in mice overexpressing dnRARα in CD11c+ cells. This indicates that vitamin A signaling in CD11c+ cells is crucial for the development of Notch2 dependent CD4+ DCs.

Figure 2. Vitamin A promotes the development of ESAM-expressing CD4+ and DN DCs. Representative histograms showing ESAM expression in CD4+, CD8+ and DN DCs in CD11c-CrenegdnRARα (black line) and CD11c‑CreposdnRARα (grey line) mice. Live, non‑autofluorescent CD11chigh CD4+, CD8+ and CD4-CD8- (DN) cells were gated for analysis. Grey filled graphs represent fluorescence minus one control. Data are representative of 6 mice and from one experiment representative of 2 performed.

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% o

f max

ESAM

CD11c-CreposdnRARα

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While abrogation of vitamin A signaling in CD11c+ cells did not influence the numbers of DN DCs in the spleen, expression levels of ESAM in DN DCs were affected in mice that overexpress dnRARα. All DN DCs in CD11c-CreposdnRARα mice expressed low levels of ESAM, while both ESAMhigh and ESAMlow DN DCs were present in control mice, as we previously observed in vitamin A deficient mice. Together, this indicates that vitamin A signaling in CD11c+ cells is required for the development of both Notch2 dependent CD4+ and DN DCs in the spleen.

MARCO+SIGNR1+ macrophages require intrinsic vitamin A signalingWe previously showed that the development of MARCO+SIGNR1+ macrophages is negatively regulated by vitamin A. On the other hand, the development of CD169+ macrophages and MARCO+SIGNR1- macrophages appear to be independent of vitamin A (chapter 3 and 6).

To determine whether intrinsic vitamin A signaling is required for the development of MZMs, we determined CD11c expression in MARCO+SIGNR1+ and MARCO+SIGNR1- macrophages. Interestingly, while MARCO+SIGNR1+ expressed low levels of CD11c, MARCO+SIGNR1+ macrophages did not express this integrin (figure 3A). CD169+ macrophages also express CD11c, although at low levels (chapter 6). Consequently, in CD11c-CreposdnRARα mice, dnRARα will be expressed in MARCO+SIGNR1+ and CD169+ macrophages, but not in MARCO+SIGNR1- macrophages. Therefore, our aim was to determine whether intrinsic vitamin A signaling is required for the development of MARCO+SIGNR1+ and CD169+ macrophages.

Spleen sections of CD11c-CreposdnRARα and CD11c-CrenegdnRARα mice were stained for MARCO and SIGNR1. In spleens of control mice, both MARCO+SIGNR1+ and MARCO+SIGNR1- macrophages were observed. However, in mice overexpressing dnRARα in all CD11c+ cells, an expansion of MARCO+SIGNR1+ macrophages was observed (figure 3B). This is consistent with vitamin A deficient mice in which also an increase of MARCO+SIGNR1+ macrophages in the splenic marginal zone was detected. However this expansion observed in immunofluorescence stainings was not reflected in mRNA expression levels. We did not detect significant differences in SIGNR1 mRNA expression in the total spleen of CD11c-CreposdnRARα mice that overexpress dnRARα

Figure 3. Development of MZM macrophages is stimulated in the absence of vitamin A signaling. (A) Representative histograms of CD11c expression in living Tim4+MARCO+SIGNR1+ (left) and Tim4+MARCO+SIGNR1- (right) macrophages in the spleen of WT mice. (B) Spleen sections of CD11c-CrenegdnRARα and CD11c‑CreposdnRARα mice were stained for MARCO (red) and SIGNR1 (blue). Representative pictures were selected of 6 mice analyzed per group. (C) Relative SIGNR1 expression was determined by quantitative PCR on total spleen homogenates of CD11c-CrenegdnRARα and CD11c-CreposdnRARα mice. Expression was normalized to the expression of housekeeping gene HPRT. (D) Spleen sections of CD11c-CrenegdnRARα and CD11c‑CreposdnRARα mice were stained for MARCO (red), SIGNR1 (blue) and CD169 (green). Representative pictures were selected of 6 mice analyzed per group. (E) Total numbers of MZ B cells were determined in CD11c‑CrenegdnRARα and CD11c‑CreposdnRARα mice. Live, non-autofluorescent, B220+CD21hiCD23- cells were gated. Numbers are given as the percentage of total live non-autofluorescent cell population. Data are shown as mean + SEM and are obtained from 1 or 2 experiments with 3 (A) or 6 (B-E) mice per group. Student T-test tested significance.

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when compared to CD11c-CrenegdnRARα mice, while MARCO mRNA expression was even significantly decreased (figure 3C). However, since DCs can also express MARCO, the mRNA expression levels in total spleens may not adequately reflect the expression levels in the MZM 34,35. Consistent with vitamin A deficient mice, CD169+ macrophages were not affected in CD11c-CreposdnRARα mice that express dnRARα in CD11c+ cells (figure 3D).

Taken together, this indicates that vitamin A signaling in MARCO+SIGNR1+ macrophages negatively regulates their development. This is in contrast to CD169+ macrophages, which develop independently of vitamin A signaling.

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Marginal zone B cells develop independently of vitamin A signaling in CD11c+ cellsVitamin A is required for the development of marginal zone B cells (MZ B cells) (29 and chapter 3). However, it remains to be elucidated whether vitamin A directly acts on these MZ  B  cells or whether other cells provide a vitamin A dependent signal that stimulates MZ  B  cell development. Previous studies have indicated that the marginal zone macrophages (MZMs) and MZ B cells are mutually dependent. However, we found an inverse correlation between MZ B cells and MZMs in vitamin A deficient mice. Since MZ B cells abundantly express RARα1 and RARα2, they are likely to respond to vitamin A directly 29. We assessed MZ B cell development in CD11c-CreposdnRARα mice that express dnRARα in CD11cpos cells, to determine whether vitamin A signaling in CD11c+ DCs and macrophages could affect the development of MZ B cells. Since MZ B cells do not express CD11c (data not shown), dnRARα is not expressed in these cells. Overexpression of dnRARα in CD11c+ cells and MZM did not affect the development of MZ B cells (figure 3E), even though MZMs were expanded in CD11c-CreposdnRARα mice. Taken together, our studies indicate that MZ B cell development is not dependent on vitamin A signaling in CD11c+ cells. Most likely, vitamin A acts directly on MZ B cells and will thereby affect their development.

DiscussionRecently, we showed that vitamin A is required for the development of Notch2 dependent CD4+ and CD4-CD8- (double negative (DN)) dendritic cells (DCs) in the spleen 28. Furthermore, our studies revealed that the development of marginal zone macrophages (MZMs) was negatively regulated by vitamin A (chapter 3).

To determine whether intrinsic vitamin A signaling is required for the development of CD4+ and DN DCs, we assessed DC development in mice that express a dominant negative RARα mutant in all CD11c+ cells (CD11c-CreposdnRARα) that results in DC specific inhibition of vitamin A signaling. Comparable to vitamin A deficient mice, the total cDC population was reduced in CD11c-CreposdnRARα mice. This DC reduction was caused by a selective decrease in CD4+ DCs, while the DN DC population was not affected. However, vitamin A signaling was essential for the ESAM expression on DN DCs as we also observed in vitamin A deficient mice. While both ESAMhighDN DCs and ESAMlowDN DC were present in control mice, in mice overexpressing dnRARα in CD11c+ cells, all DN DCs expressed low levels of ESAM. These results indicate that intrinsic vitamin A signaling is required for the development of both Notch2 dependent ESAMhighCD4+ and ESAMhighDN DCs.

The mechanism via which vitamin A regulates the development of Notch2 dependent DCs remains to be elucidated. Various transcription factor knockout mice have been generated which showed a reduction in one or multiple DC subsets. For example, RelB, IRF2 and IRF4 knockout mice exhibited a reduction in both CD4+ and DN DCs 36-

38. The expression of one of these transcription factors could potentially be vitamin A dependent. However, we did not observe effects of RA on the expression of these transcription factors in preliminary studies. Also, other pathways that are involved in DC

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development could be vitamin A dependent, such as LTαβ, EBI2, and Notch signaling. We observed specific reduction of Notch2-dependent DCs when vitamin A signaling was inhibited. Notch2 signaling in MZ B cells is regulated via by proteolytic activity of ADAM10 (a disintegrin and metallopeptidase 10) 39 and ADAM10 expression has been shown to be vitamin A dependent 40. If vitamin A regulates ADAM10 expression in murine DCs or its precursors, this could explain their vitamin A dependence. Very recently, the G coupled protein receptor Epstein-Barr virus induced molecule 2 (EBI2) was shown to be essential for positioning and homeostasis of CD4+ DCs in the splenic bridging channels 41,42. This is presumably mediated by the high concentrations of EBI2 ligands (7α,25-dihydroxycholesterol (7α,25-OHC)) in this region 41,43. Vitamin A could potentially regulate the EBI2 receptor, but this is still unclear.

The developmental pathway of marginal zone macrophages (MZMs) in the spleen remains largely unclear. Growth factor M-CSF (also known as CSF-1) is required for their development 44. Recently the nuclear receptor (LXRα) was demonstrated to be required for the differentiation of both CD169+ and SIGNR1+ macrophages in the marginal zone 45. In chapter 3, we showed that MZM development is negatively regulated by vitamin A: an expansion of SIGNR1+ macrophages was observed in vitamin A deficient mice, while RA supplementation significantly reduced SIGNR1+ macrophages. An expansion of CD11c+MARCO+SIGNR1+ macrophages was also observed in spleens of CD11c-CreposdnRARα mice in which RA signaling was abrogated. Thus, MZM-intrinsic vitamin A signaling inhibits the development of MARCO+SIGNR1+ MZMs in the spleen in a cell autonomous manner. Vitamin A signaling is mediated via retinoic acid binding to the RAR and RXR nuclear receptors that subsequently bind to retinoic acid response elements (RARE) in the promoter regions. RXR also forms heterodimers with LXR, which bind to LXR response elements. Possibly that increased or decreased presence of retinoic acid may affect LXR regulated gene transcription, but this remains to be investigated 45-47.

Previous reports have indicated a mutual dependence between MZMs and MZ B cells 18,48-50. However, in vitamin A deficient mice, we observed an inverse correlation between MZ B cells and MZMs, as in these mice MZ B cells were decreased and MZMs were increased. In CD11c-CreposdnRARα mice, which have an impaired vitamin A signaling in MZMs but not in MZ  B  cells, we observed an increased MZMs population, while MZ B cells were not affected. This indicates that the effects of vitamin A on MZ B cells and MZMs are independent of the mutual dependency between MZMs and MZ B cells that was previously reported.

In conclusion, our studies demonstrate that vitamin A signaling significantly affects the development of macrophages and DCs in the marginal zone in the spleen in a cell autonomous manner. Together with our previous studies that revealed the effects of dietary vitamin A deficits on these macrophages and DCs, our studies indicate that deficiencies in vitamin A levels may have important consequences for proper development and functioning of the systemic immune system.

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