Constitutive Expression of TNF-Related Activation-Induced Cytokine (TRANCE)/Receptor Activating NF-κB Ligand (RANK)-L by Rat Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDCs) are a subset of DCs whose major function relies on their capacity to produce large amount of type I IFN upon stimulation via TLR 7 and 9. This function is evolutionary conserved and place pDC in critical position in the innate immune response to virus. Here we show that rat pDC constitutively express TNF-related activation-induced cytokine (TRANCE) also known as Receptor-activating NF-κB ligand (RANKL). TRANCE/RANKL is a member of the TNF superfamily which plays a central role in osteoclastogenesis through its interaction with its receptor RANK. TRANCE/RANK interaction are also involved in lymphoid organogenesis as well as T cell/DC cross talk. Unlike conventional DC, rat CD4high pDC were shown to constitutively express TRANCE/RANKL both at the mRNA and the surface protein level. TRANCE/RANKL was also induced on the CD4low subsets of pDC following activation by CpG. The secreted form of TRANCE/RANKL was also produced by rat pDC. Of note, levels of mRNA, surface and secreted TRANCE/RANKL expression were similar to that observed for activated T cells. TRANCE/RANKL expression was found on pDC in all lymphoid organs as well blood and BM with a maximum expression in mesenteric lymph nodes. Despite this TRANCE/RANKL expression, we were unable to demonstrate in vitro osteoclastogenesis activity for rat pDC. Taken together, these data identifies pDC as novel source of TRANCE/RANKL in the immune system

TRANCE mRNA expression on DC and pDC subsets.

<p><b>A.</b> The expression of TRANCE mRNA was assessed in total mRNA prepared from FACS-sorted spleen cDCs and pDCs (n = 3) and from lymph node cells (n = 3) before and after activation with ConA. <b>B.</b> Gating strategy for sorting pDC subsets. After positive selection of 85C7⁺ cell, cells were stained with anti-CD45R and anti-CD4 mAbs, then sorted as 85C7⁺CD45R⁺CD4^high and CD4^low cells (dot plots, before FACS-sorting). Purity was routinely >99.5% for both population (Dot plots, after FACS-sorting). Sorted pDC subsets were also stained with anti-Siglec-H Ab (histrograms, after FACS-sorting). <b>C.</b> The expression of TRANCE mRNA was assessed in CD4⁺ and CD4⁻ subsets of pDCs at resting state and after maturation in presence of either CpG A or B. The results are expressed as a ratio of TRANCE to HPRT mRNA expression and represent the mean ± SD of three independent experiments. *, p<0,05; **, p<0,01; ***, p<0.001.</p

TRANCE expression on pDC in various lymphoid organs.

<p><b>A.</b> Freshly prepared spleen cells were stained with various antibodies to identify CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), B cells (CD45R⁺OX33⁺), NK cells (NKR-P1A ^highCD3⁻), CD11b/c⁺ cells, cDC (CD103⁺), pDC (85C7⁺ CD4⁺) together with RANK-Fc (bold histogram) or the secondary Ab alone (dotted histogram). <b>B.</b> Cell suspensions from various lymphoid organs were stained with 85C7 and CD4 antibodies to identify CD4^high pDC and RANK-Fc (bold histogram) or the secondary Ab alone (dotted histogram). Similar results were obtained in 3 independent experiments for each panel.</p

Soluble TRANCE production by pDCs.

<p>Freshly sorted splenic pDC (n = 4) and cDC (n = 3) were stimulated in complete RPMI with CpG B or A (5 µM). Lymph node cells were stimulated with ConA (5 µg/mL). Supernatants were collected at 6 and 24 h and soluble TRANCE was assessed by ELISA (R&D systems). The detection threshold of the ELISA was 24 pg/mL. ***, p<0.001.</p

TRANCE protein expression on spleen DC subsets.

<p>Freshly isolated (upper histograms) or 24 h CpG B-stimulated (lower histrograms) CD4⁺ cDCs, CD4⁻ cDCs and CD4^high pDC or were stained with RANK-Fc (bold histrogram) or control Ig (dotted histogram).</p

Kinetics of TRANCE protein expression on activated pDCs.

<p>FACS sorted CD4^high (upper panels) and CD4^low (lower panels) pDC were stimulated with CpG B and stained stained with RANK-Fc (bold lines) or the secondary Ab alone (thin lines) at 0 (resting), 6, 24 and 48 h after stimulation. Similar results were obtained in 3 independent experiments.</p

pDCs inhibits in vitro osteoclastogenesis.

<p>Bone marrow cells were differentiated in osteoclast and stained for their expression of tartrate-resistant acid phosphatase (TRAP) (Leukocyte Acid Phosphatase Assay kit; Sigma). Soluble TRANCE was used 100 ng/mL as a positive control for the induction of osteoclasts. When indicated, 2×10⁵ FACS sorted CD4^high pDC were added to bone marrow cells at day 0 in addition to TRANCE. pDCs were shown to inhibit TRANCE-mediated osteoclastogenesis in these conditions. These results are representative of 5 independent experiments.</p

E2-2 and IFNα expression by TRANCE+ and − pDCs.

<p><b>A.</b> Gating strategy for isolating TRANCE⁺ and ⁻ spleen pDC. After positive selection of 85C7⁺ cells (left dot plot), cells were stained with CD45R mAb and Rank-Fc. TRANCE⁻ and TRANCE⁺ 85C7⁺CD45R⁺ cells were sorted on a FACS Aria (middle dot plot). Purity was >98% for both population (right dot plots). <b>B.</b> E2-2 mRNA expression was assessed by Q-PCR in FACS sorted TRANCE+ (TR+) and – (TR−) population as well as in splenic cDCs (n = 2). <b>C.</b> The expression of IFNα mRNA was assessed by Q-PCR in FACS-sorted population (n = 3) as well as in 85C7⁺CD45R⁺ cells (total pDC), at resting state and after 6 h activation with CpG 2216 (5 µM). The results are expressed as a ratio of IFNα to HPRT mRNA expression and represent the mean ± SD of two or three independent experiments.</p

Surface TRANCE protein detection.

<p><b>A.</b> Freshly FACS sorted CD4^high pDC were stained with a polyclonal antibody to TRANCE, OPG-Fc or RANK-Fc. <b>B.</b> Resting and Con-A-activated lymph node cells were stained with RANK-Fc and used as a positive control. <b>C.</b> RANK-Fc was preincubated with soluble TRANCE for 20 minutes at 4°C before staining pDC as in A.</p