Unique lamina propria stromal cells imprint the functional phenotype of mucosal dendritic cells

Abstract

Mucosal dendritic cells (DCs) in the intestine acquire the unique capacity to produce retinoic acid (RA), a vitamin A metabolite that induces gut tropism and regulates the functional differentiation of the T cells they prime. Here, we identified a stromal cell (SC) population in the intestinal lamina propria (LP), which is capable of inducing RA production in DCs in a RA- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent fashion. Unlike DCs, LP SCs constitutively expressed the enzymatic machinery to produce RA even in the absence of dietary vitamin A, but were not able to do so in germ-free mice implying regulation by microbiota. Interestingly, DCs promoted GM-CSF production by the SCs indicating a two-way cross-talk between both cell types. Furthermore, RA-producing LP SCs and intestinal DCs localized closely in vivo suggesting that the interactions between both cell types might have an important role in the functional education of migratory DCs and therefore in the regulation of immune responses toward oral and commensal antigens.

Figure 1. A subset of LP SCs have the capacity to produce RA

a Analysis of RALDH activity using aldefluor in single-cell suspensions prepared from SI LP and assessed by flow cytommetry for the indicated markers. Left lower panel displays CD45⁻aldefluor⁺ cells (gate R1, black color) within the whole SI LP SC population (grey color). Data shows a representative experiment of more than 5 independent experiments. b mRNA levels of the RA producing enzymes RALDH1 (aldh1a1) RALDH2 (aldh1a2) and RALDH3 (aldh1a3) were measured by qRT-PCR in sorted CD45⁻Epcam⁻CD31⁻Pdpn⁺ cells (SCs, black bars) and CD45⁻Epcam^hi cells (IEC, white bars). Data shown correspond with RNA from three independent experiments that were quantified at the same time. c Supernatants from sorted SCs were used to stimulate F9-RARE-lacz cells and β galactosidase production by these cells measured using a colorimetric reaction. Left panel compares the absorbances obtained when F9-RARE-lacz cells were left untreated (black bar) or supernatants were added to the culture (white bar), right panel shows an estimation of the RA cc present in the supernatants. Represented is the average+/−s.d. from three independent experiments pooled together (p=0.0269 t-test non paired). d Aldefluor staining of CD45⁻ Pdpn⁺ stromal cells was compared between specific pathogen free (SPF) and germ free mice (GF) or e between vitamin A sufficient (VAS) and vitamin A (VAD) mice. Shown is a representative experiment out of three independent experiments (p = 0.18 n.s. t-test non paired).

Figure. 2. Aldefluor positive SCs are abundant within the intestinal lamina propria and locate closely to intestinal DCs

a–c Confocal imaging of aldefluor stained live SI explants after IEC removal. Aldefluor staining was followed by immunofluorescence with the indicated antibodies, anti-CD31 (a), anti-CD11c (b) or anti-CD11c plus anti-podoplanin (b, right panel) or plus anti-CD103 (c, right panel). In the indicated images (b, central panel) explants were stained with aldefluor in the presence of the RALDH inhibitor DEAB. White arrowheads point out aldefluor⁺ DCs in close contact with aldefluor⁺ SCs, yellow arrowheads indicate podoplanin⁺ aldefluor⁺ cells. Quantification shows the percentage of CD11c⁺ CD103⁺ cells in direct contact with aldefluor bright LP cells from more than ten different images.

Figure 3. DCs require RAR signaling to induce RA producing enzymes when cultured with Pdpn⁺ CD31⁻ SI LP SCs

a Aldefluor staining of splenic DCs (gated CD45⁺MHCII^hiCD11c⁺) after 24h culture alone (Untreated) or together with SI SCs (SCs cond.). DEAB (100 µM) was added at the time of the staining together with aldefluor where indicated. Data show one representative experiment of more than 5 experiments with identical results. b FACs analysis of IL-17A (left panel) or α4β7(right panel) in PMA/Ionomycin re-stimulated OTII cells primed with splenic DCs for 4–5 days. When indicated, DEAB (10 µM) was added to the DC-T cell culture and maintained during the whole length of the experiment. Analysis shows the result from one representative experiment of at least two experiments with similar results. c mRNA levels of the RA producing enzymes RALDH1 (aldh1a1) RALDH2 (aldh1a2) and RALDH3 (aldh1a3) were measured by qRT-PCR in splenic DCs after 24hr culture in media alone or media supplemented with SC supernatants (SCs sups, obtained as in Fig1a). Data shown correspond to RNA from three independent experiments that were quantified at the same time. d Aldefluor staining of splenic DCs (gated CD45⁺MHCII^hiCD11c⁺) after 24h culture alone or together with sorted SCs (SCs cond, left panel) or their supernatants (SCs sups, right panel). The global RAR global antagonist BMS204493 (RAR inh) was added to the culture at concentration of 20nM or otherwise indicated. Data shown are from one representative experiment of at least three experiments.

Figure 4. GM-CSF production by stromal cells is enhanced by DCs and required for the imprinting of DCs with high RALDH activity

a Aldefluor staining of splenic DCs after 24 h treatment with SI LP CD45⁻Epcam⁻Pdpn⁺CD31⁻ stromal cells (SCs) or with media supplemented with increasing concentrations of RA. Data from one representative experiment of three experiments with similar results b Left panel shows aldefluor staining of a cell line established from sorted primary CD45⁻Pdpn^hi splenic cells (Sp SCs) versus SI LP CD45⁻Epcam⁻Pdpn⁺CD31⁻ stromal cells (SCs). Middle panel shows aldefluor staining of splenic DCs (CD45⁺CD11c⁺MHCII^hi gate) cultured for 24h with the aforementioned cell line in the presence or not of RA (100 nM). Right panel shows aldefluor staining of splenic DCs treated with RA plus concentrated sups from the aforementioned cell line. Sups were digested with proteinase K when indicated. Shown is the data from one representative experiment of two. c left panel shows aldefluor staining of splenic DCs after 24h treatment with RA plus sequential fractions of the sups from Sp SCs (fractionation details within the material and methods section). Right panel shows an estimate of the size of the proteins contained within the fraction that gave us the most activity. d left panel shows aldefluor staining of splenic DCs cultured for 24 hours alone or together with SCs from WT or GM-CSF KO mice. Anti-GM-CSF blocking antibody was added where indicated. Right panel shows the fold increase on aldefluor staining (mean fluorescence intensity, MFI) on splenic DCs co-cultured with SI LP CD45⁻Epcam⁻Pdpn⁺CD31⁻ stromal cells (SCs) from WT vs GM-CSF KO mice. Data shows the results from three independent experiments * P < 0.05 (unpaired t-test) e Aldefluor staining of splenic DCs after 24h culture with SI LP CD45−Epcam−Pdpn+CD31− stromal cells (SCs) in the same well or separated by a permeable membrane (transwell) Bar graphs shows data from three replicate wells within one experiment. Data shows one representative experiment out of two. f shows GM-CSF by intracellular staining (left panel) or ELISA (right) on SI LP CD45−Epcam−Pdpn+CD31− stromal cells (SCs) cultured for 24h alone or together with splenic DCs. Left panel shows one representative experiment out of three. Right panel shows data analyzed corresponding to five independent experiments ** P <0.01 (unpaired t-test)

Figure 5. CD11b⁺CD103⁺ DC numbers and RA producing capacity are diminished in GM-CSF −/− mice

a Upper row shows CD11b vs CD103 staining of SI LP DCs (gated as CD45⁺ CD11c^hi MHCII^high) in WT vs GM-CSF KO mice. Lower row shows the percentage of the indicated populations among the total number of SI LP cells in WT vs GM-CSF KO mice, left panel total CD103⁺ DCs, middle panel CD11b⁺CD103⁺ DCs and right panel CD11b⁻CD103⁺ DCs. b Aldefluor staining of the CD11b⁺ (upper row) and CD11b⁻ (lower row) SI LP CD103⁺ DC population in WT vs GM-CSF KO mice indicating the percentage of aldefluor high cells (middle panel) as well as the aldefluor mean fluorescence intensity (MFI) for the whole population (right panel). Data shows one representative experiment of more than three performed (n.s. P>0.05, * P<0.05, ** P<0.001, *** P<0.0005)