Figure 1.

Nod2 signaling is required in BM cells to reconstitute the mo-DCs pool in the peritoneal cavity despite similar monocyte recruitment. (A) Experimental protocol. Mixed BM chimeras were generated by transferring WT (expressing CD45.1)(blue) and Nod2^-/- (expressing CD45.2) (red) cells in a 1:1 ratio into lethally irradiated Nod2-deficient recipients. Cells were isolated from the blood and the peritoneum 8 weeks after reconstitution. (B) Peritoneal cells were harvested and the proportion of mo-Mac (MHCII^-ICAM2⁺) and mo-DCs (MHCII⁺CD226⁺) was determined by flow cytometry by gating within the CD115⁺CD11b⁺ cells. The frequency of the CD226⁺ DC is calculated within the CD115⁺ CD11b⁺ cells. (C) The ratio of mo-DCs/mo-Macs from frequencies obtained in B (n=4 mice). (D) Reconstitution index of Ly6C⁺ monocytes in the colon (CD11c^-CD11b⁺Ly6C⁺CCR2⁺ gate) divided by the same ratio in the blood (n=6 mice). (E) 8 weeks after reconstitution, mixed chimera mice were treated with a 5-day course of 2% DSS (n=7 mice)(E-G). (E) Monocytes content (in the blood: CD11b⁺ Ly6hi⁺, in the colon: CD11c^-CD11b⁺Ly6C⁺CCR2⁺MHCII^-) in the blood (CD11b⁺ Ly6hi⁺) of untreated and blood and colonic lamina propria cells (cLP) in DSS-treated mice. (F) Ratio of WT and Nod2-/- monocyte frequencies (colon/blood). (G) Colonic cells were analyzed as described in Supplementary Figure 2A . Numbers per million of monocytes MHCII⁺ (CD11c^-CD11b⁺Ly6C⁺CCR2⁺MHCII⁺), mo-DCs (CD11c⁺CD11b⁺Ly6C⁺CCR2⁺MHCII⁺), mo-Macs (CD11c^-CD11b⁺Ly6C^-CCR2^-MHCII⁺), monocytes MHCII^- (CD11c-CD11b⁺Ly6C⁺CCR2⁺MHCII^-), CD11c⁺Macs (CD11c⁺CD11b⁺Ly6C⁺CCR2^-MHCII⁺), DC1 (CD11c⁺CD11b^-) and DC2 (CD11c⁺CD11b⁺Ly6C^-CCR2^-MHCII⁺) in the cLP (number of cells per million of live cells). The ratio of mo-DCs/mo-Mac from total cell number. Bars indicate mean ± SEM. Statistical significance was assessed by the non-parametric Mann-Whitney U test. ns, non significant.