Origin of the lamina propria dendritic cell network

Abstract

CX(3)CR1(+) and CD103(+) dendritic cells (DCs) in intestinal lamina propria play a key role in mucosal immunity. However, the origin and the developmental pathways that regulate their differentiation in the lamina propria remain unclear. We showed that monocytes gave rise exclusively to CD103(-)CX(3)CR1(+) lamina propria DCs under the control of macrophage-colony-stimulating factor receptor (M-CSFR) and Fms-like thyrosine kinase 3 (Flt3) ligands. In contrast, common DC progenitors (CDP) and pre-DCs, which give rise to lymphoid organ DCs but not to monocytes, differentiated exclusively into CD103(+)CX(3)CR1(-) lamina propria DCs under the control of Flt3 and granulocyte-macrophage-colony-stimulating factor receptor (GM-CSFR) ligands. CD103(+)CX(3)CR1(-) DCs but not CD103(-)CX(3)CR1(+) DCs in the lamina propria constitutively expressed CCR7 and were the first DCs to transport pathogenic Salmonella from the intestinal tract to the mesenteric lymph nodes. Altogether, these results underline the diverse origin of the lamina propria DC network and identify mucosal DCs that arise from pre-DCs as key sentinels of the gut immune system.

Figure 1. Characterization of phenotypically distinct DC subsets in the small bowel

A. Single cell suspensions prepared from different anatomical compartments of the small bowel (SB) including the IEC fraction, lamina propria (LP), serosa with the intestinal muscularis and PP were analyzed by seven color flow cytometry. The results were compared to the whole SB depleted of PP (SB without (w/o) PP). To define CD11c^hi and CD11c^lo DCs, gates were set on viable DAPI⁻CD45⁺MHCII^hi cells. CD11c^hi DCs were further divided into CD103⁺CD11b⁻, CD103⁺CD11b⁺ and CD103⁻CD11b⁺ DCs as indicated in the dot plots. Dot plots in the left column show the % of CD11c^hi and CD11c^lo DCs among total DAPI⁻CD45⁺MHCII^hi cells. Dot plots in the right column show the % of CD103⁺CD11b⁻, CD103⁺CD11b⁺ and CD103⁻CD11b⁺ DCs among total MHCII^hiCD11c^hi or MHCII^hiCD11c^lo (serosa and muscularis) DCs. B. Images show the distribution of CD103⁺CD11c-YFP⁺ cells in the villi of distal SB from CD11c-EYFP transgenic mice. Magnification 400x. Scale bar 10 nm. C. Dot plots show the % of CD103⁺CD11b⁻, CD103⁺CD11b⁺ and CD103⁻-CD11b⁺ DCs among CD45⁺MHCII^hiCD11c^hi cells in the SB depleted of PP in WT mice and in the total SB of Id2^−/− mice. D. Overlay histograms show the differential MHCII, CD8α, CX3CR1, M-CSFR and F4/80 expression among each intestinal DC subset. E. Images show purified CD103⁺CD11b⁺ and CD103⁻CD11b⁺ SB LP DCs spun onto glass slides and stained with Giemsa. Magnification 600x. Scale bar 10 nm. F. CFSE labeled OTII cells were cultured with purified CD103⁺CD11b⁺ or CD103⁻CD11b⁺ SB LP DCs, splenic DCs or splenic B cells pulsed with OVA-peptide. Numbers are the % of OTII cells that have not proliferated in each group (representative data of two independent experiments done in triplicates).

Figure 2. M-CSFR, Flt3 and GM-CSFR control the development of LP DCs

A. Dot plots show the % of CD103⁺CD11b⁻, CD103⁺CD11b⁺ and CD103⁻CD11b⁺ cells among DAPI⁻CD45⁺MHCII^hiCD11c^hi SB LP DCs in M-CSFR^−/−, Flt3^−/− and GM-CSFR^−/− mice (right panels) or control WT littermates (left panels). PP were excised from the SB of Flt3^−/−, GM-CSFR^−/− and control littermates, but not from the SB of M-CSFR^−/− and their MCSFR^+/+ control littermates. B. Bar graphs show the relative change of absolute cell counts among SB LP DC subsets in M-CSFR^−/−, Flt3^−/− and GM-CSFR^−/− mice compared to WT mice. Error bars represent mean +/− SD from 4 (M-CSFR^−/−) to 6 (Flt3^−/− and GM-CSFR^−/−) combined experiments. (*) − 0.05>p>0.005, (**) − p<0.005. C. WT CD45.1⁺ mice were lethally irradiated and reconstituted with a mixture of 1:1 CD45.1⁺ WT and CD45.2⁺ Flt3^−/− or GM-CSFR^−/− BM cells or with a mixture of 1:10 CD45.1⁺ WT and CD45.2⁺ MCSFR^−/− fetal liver cells. Bar graphs show the % of CD45.2⁺ KO and CD45.1⁺ WT cells among blood cells (granulocytes or monocytes) used as a control and each SB LP DC subset. Error bars represent means +/− SD from 3 simultaneously analyzed experiments. (*) − 0.05>p>0.005, (**) − p<0.005 as compared to the chimerism of blood granulocytes or monocytes.

Figure 3. Origin of LP DCs

A. Untreated (No DT) CD45.2⁺ CD11c-DTR recipients were injected i.v. with MDP, CDP, pre-DC or Ly6C^hi monocytes (mono-) purified from the BM of CD45.1⁺ WT mice. Dot plots show the % of donor-derived CD45.1⁺ cells among each SB LP DC subset seven days after adoptive transfer and represent three to four independent experiments (n=1 to 2). B. Turnover of LP DCs in parabiotic mice. Each parabiotic pair consisted of a CD45.1⁺ and CD45.2⁺ WT mouse with shared blood circulation. LP DC chimerism was analyzed two weeks after initiation of parabiosis. Graph shows the % of donor parabiont-derived cells among SB LP DC subsets in a recipient parabiont. C. Origin of LP DCs in DC-depleted mice. Twelve h after DT administration, CD45.2⁺ CD11c-DTR chimeric mice were injected i.v. with MDP, CDP or Ly6C^hi monocytes and analyzed as described in (A). D. Tracing the origin of LP DCs in reporter mice. Graphs show the % of eGFP⁺ cells among SB LP DC subsets in LysM-Cre × Rosa26-stop^floxEGFP mice.

Figure 4. Migration of the LP DC subsets to the MLN in the steady state

A. Dot plots show the % of CD103⁺CD11b⁻, CD103⁺CD11b⁺ and CD103⁻CD11b⁺ DCs among DAPI⁻CD45⁺MHCII^hiCD11c⁺ MLN DCs (left panel). Histograms show the CX3CR1 expression levels on gated CD103⁻CD11b⁺ MLN DCs compared to CD103⁻CD11b⁺ SB LP DCs isolated from CX3CR1^GFP/+ knock-in mice. B. Dot plots show the % eGFP⁺ cells among total CD103⁺CD11b⁻, CD103⁺CD11b⁺ and CD103⁻CD11b⁺ SB LP DCs (left column) and MLN DCs (right column) in LysM-Cre × Rosa26-stop^floxEGFP mice. Data represent one of 3 independent experiments. C. Bar graphs show CCR7 mRNA relative expression units (RU) in purified CD103⁺CD11b⁺ and CD103⁻CD11b⁺ SB LP DCs compared to purified splenic DCs from naive WT mice. Bar graph shows the mean ± SD of data from four independent cell purifications tested in the same qPCR reaction. D, E. Dot plots and bar graph show the relative (D) and absolute (E) numbers of CD103⁺CD11b⁻, CD103⁺CD11b⁺ and CD103⁻CD11b⁺ MLN DCs in WT and CCR7^−/− mice. Data represent the mean ± SD of 4 combined experiments.

Figure 5. CD103⁺CD11b⁺ DCs are the first DCs to transport Salmonella Typhimurium to the MLN after oral infection

A–C. Mice were infected with Salmonella either orally (per os, p/o) or intraperitoneally (i/p). 18 h (i/p) or 24 h (p/o) post-infection, MLNs were collected and MLN single cell suspensions were stained for DC surface markers and intracellular Salmonella CSA and subjected to flow cytometry. A. Dot plots show the % of Salmonella⁺ cells among each MLN DC subset after p/o or i/p infection compared to uninfected controls. B. Bar graph shows the absolute number of Salmonella⁺ (SAL⁺) cells among each MLN DC subset 24 h after oral infection. Data represent the mean ± SD of 3 independent experiments. C. Images show S Salmonella ⁺ cells in purified CD103⁺CD11b⁺ but not CD103⁺CD11b⁻ and CD103⁻CD11b⁺ MLN DC 24 h after oral infection. Peritoneal macrophages (Per. Mφ) isolated 4 h after i/p infection, were used as a positive control. Magnification 600x. Scale bar 10 nm. D–E. 24 h after oral infection with Salmonella, SB were isolated and stained for DC surface markers and intracellular Salmonella antigens and analyzed by flow cytometry. D. Dot plots show the % of Salmonella⁺ cells among each LP SB DC subset after oral (per os, p/o) infection compared to uninfected controls. E. Images show Salmonella⁺ cells among purified SB LP CD103⁺CD11b⁺ and CD103⁻ CD11b⁺ but not CD103⁺CD11b⁻ DCs after oral infection. Magnification 600x. Scale bar 10 nm.

Figure 6. Transport of Salmonella Typhimurium to the MLN is impaired in Flt3^−/− mice

A. Graphs show the numbers of Salmonella colony forming units (CFU) recovered from the homogenized terminal SB and total MLN of WT and Flt3^−/− mice 2 days after oral Salmonella infection. Data represent the results of 3 independent experiments. B. Graphs show Salmonella CFU recovered from the homogenized total MLN of WT and CCR7^−/− mice 2 days after oral Salmonella infection. Data represent the results of 3 independent experiments.