E-NPP3 controls plasmacytoid dendritic cell numbers in the small intestine

<div><p>Extracellular adenosine 5’-triphosphate (ATP) performs multiple functions including activation and induction of apoptosis of many cell types. The ATP-hydrolyzing ectoenzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) regulates ATP-dependent chronic allergic responses by mast cells and basophils. However, E-NPP3 is also highly expressed on epithelial cells of the small intestine. In this study, we showed that E-NPP3 controls plasmacytoid dendritic cell (pDC) numbers in the intestine through regulation of intestinal extracellular ATP. In <i>Enpp3</i>^-/- mice, ATP concentrations were increased in the intestinal lumen. pDC numbers were remarkably decreased in the small intestinal lamina propria and Peyer’s patches. Intestinal pDCs of <i>Enpp3</i>^-/- mice showed enhanced cell death as characterized by increases in annexin V binding and expression of cleaved caspase-3. pDCs were highly sensitive to ATP-induced cell death compared with conventional DCs. ATP-induced cell death was abrogated in <i>P2rx7</i>^-/- pDCs. Accordingly, the number of intestinal pDCs was restored in <i>Enpp3</i>^-/- <i>P2rx7</i>^-/- mice. These findings demonstrate that E-NPP3 regulates ATP concentration and thereby prevents the decrease of pDCs in the small intestine.</p></div

Enhancement of ATP-induced apoptosis of pDCs.

<p><b>(A, B)</b> Cells isolated from MLNs were treated with the indicated concentrations of ATP for 3 h. Annexin V-positive cells (A) and active caspase-3-positive cells (B) among PDCA-1⁺ CD11c^med pDCs and PDCA-1^- CD11c^high cDCs were analyzed by flow cytometry. Representative histograms are shown (left) and the means ± SD (n = 3) of the percentages of annexin V-positive (A) and active caspase-3-positive cells (B) are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. <b>(C)</b> Frequency of active caspase-3-positive cells among CD45⁺ PDCA-1⁺ CD11c^med pDCs and and PDCA-1^- CD11c^high cDCs from the PPs and SILP of mice injected with 300 μl PBS (n = 6) or 1 mM ATP-γS (n = 5) into their small intestinal lumen. Representative histograms are shown (left) and the means ± SD of the percentages of positive cells are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, NS: not significant.</p

P2X7-dependent induction of pDC apoptosis in vivo.

<p><b>(A, B)</b> Frequencies of annexin V-positive (A) and active caspase-3-positive (B) cells gated on CD45⁺ PDCA-1⁺ CD11c^med pDCs from the PPs and SILP of wild-type, <i>Enpp3</i>^<i>-/-</i>, <i>P2rx7</i>^<i>-/-</i>, and <i>Enpp3</i>^<i>-/-</i> <i>P2rx7</i>^<i>-/-</i> mice (n = 7 per groups in a, and n = 5 per groups in b). Representative histograms are shown (left) and the means ± SD of the percentages of annexin V-positive (A) and active caspase-3-positive cells (B) are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01 <b>(c)</b> Frequency and number of PDCA-1⁺ CD11c^med pDCs in the PPs and SILP from wild-type, <i>Enpp3</i>^<i>-/-</i>, <i>P2rx7</i>^<i>-/-</i>, and <i>Enpp3</i>^<i>-/-</i> <i>P2rx7</i>^<i>-/-</i> (n = 13 per groups) mice. Representative dot plots are shown (left) and the means ± SD of the percentages of pDCs are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Expression of <i>Enpp3</i> in small intestinal epithelia.

<p><b>(A)</b> Quantitative RT-PCR analysis of <i>Enpp3</i> mRNA expression in the indicated tissues (n = 3). <b>(B)</b> Quantitative RT-PCR analysis of <i>Enpp3</i> mRNA expression in epithelial cells and lamina propria in four parts of the small intestine. A smaller number represents a more proximal portion of the intestine (n = 4). <b>(C)</b> Quantitative RT-PCR analysis of <i>Enpp3</i> mRNA expression in epithelial cells of the small intestine in specific-pathogen free (SPF) and germ-free (GF) mice (n = 3). <b>(D)</b> Immunohistochemical analysis of the small intestine. E-NPP3 (red) and DAPI (blue). Swiss roll frozen sections were stained with the anti-mouse E-NPP3 antibody. A smaller number represents more proximal portion of the intestine. Scale bars, 100 μm. <b>(E)</b> ATP concentrations in luminal contents of the small intestine of wild-type and <i>Enpp3</i>^<i>-/-</i> mice. All data are mean values ± SD (n = 6 per groups). *<i>p</i> < 0.05. <b>(F)</b> ATP concentration in the proximal portion of the small intestinal lumen from wild-type, <i>Enpp3</i>^<i>-/-</i>, <i>Kit</i>^{<i>W-sh/W-sh</i>} and <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} mice. All data are mean values ± SD (n = 4 per groups). *<i>p</i> < 0.05, NS: not significant. <b>(G)</b> ATP concentration in the proximal portion of the small intestinal lumen from <i>Kit</i>^{<i>W-sh/W-sh</i>} and <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} mice with or without adoptive transfer of wild-type or <i>Enpp3</i>^<i>-/-</i> bone marrow-derived mast cells. All data are mean values ± SD (n = 5 for mast cells transferred mice groups and <i>Kit</i>^{<i>W-sh/W-sh</i>} mice group, and n = 3 for non transferred <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} mice group). *<i>p</i> < 0.05, NS: not significant.</p

P2X7-dependent induction of pDC apoptosis in vitro.

<p><b>(A)</b> Expression of P2X receptors in pDCs or cDCs of MLN was analyzed by quantitative RT-PCR. Data are means ± SD (n = 3). *<i>p</i> < 0.05, **<i>p</i> < 0.01. <b>(B)</b> mRNA expression of <i>P2rx4</i> and <i>P2rx7</i> in pDCs and cDCs of SILP, PP, bone marrow (BM) and spleen (SPL) was analyzed by quantitative RT-PCR. Data are means ± SD (n = 3). *<i>p</i> < 0.05, **<i>p</i> < 0.01. <b>(C, D)</b> Isolated MLN pDCs of wild-type and <i>P2rx7</i>^<i>-/-</i> mice were treated with the indicated concentrations of ATP for 3 h. Annexin V-positive cells (C) and active caspase-3-positive cells (D) were analyzed by flow cytometry. Representative histograms are shown (left) and the means ± SD of the percentages of annexin V-positive (C) and active caspase-3-positive cells (D) (n = 4) are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Decrease in the number of intestinal pDCs in <i>Enpp3</i>^<i>-/-</i> mice.

<p><b>(A</b>–<b>C)</b> Frequency and number of CD45⁺ PDCA-1⁺ CD11c^med pDCs (A, C) and CD45⁺ PDCA-1^- CD11c ^high cDCs (B, C) in the Peyer’s patches (PPs), small intestinal lamina propria (SILP), bone marrow (BM), and spleen (SPL) of wild-type and <i>Enpp3</i>^<i>-/-</i> mice. All data are mean values ± SD (n = 7 for PP and SILP, and n = 6 for SPL and BM). *<i>p</i> < 0.05, NS: not significant. <b>(D)</b> Frequency of CD45⁺ PDCA-1⁺ CD11c^med pDCs in the PPs and SILP of wild-type (n = 6), <i>Enpp3</i>^<i>-/-</i> (n = 8), <i>Kit</i>^{<i>W-sh/W-sh</i>} (n = 7), and <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} (n = 6) mice. All data are mean values ± SD. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, NS: not significant. <b>(E, F)</b> Frequency of annexin V-positive (E) and active caspase-3-positive (F) cells gated on CD45⁺ PDCA-1⁺ CD11c^med pDCs from the PPs and SILP of wild-type and <i>Enpp3</i>^<i>-/-</i> mice. Representative histograms are shown (left) and the means ± SD of the percentages of positive cells (right) are shown (n = 6 in e, and n = 5 in f). *<i>p</i> < 0.05, **<i>p</i> < 0.01. <b>(G)</b> Frequency of PDCA-1⁺ CD11c^med pDCs in the PPs and SILP from antibiotic-treated wild-type (n = 11) and <i>Enpp3</i>^<i>-/-</i> (n = 12) mice as well as untreated wild-type (n = 10) and <i>Enpp3</i>^<i>-/-</i> (n = 10) mice. Data are the means ± SD of the percentages of pDCs. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, NS: not significant.</p