Abstract
Fibroblastic reticular cells (FRCs) and their specialized collagen fibers termed ‘conduits’ form fundamental structural units supporting lymphoid tissues. In lymph nodes, conduits are known to transport interstitial fluid and small molecules from afferent lymphatics into the nodal parenchyma. However, the immunological contributions of conduit function have remained elusive. Here, we report that intestinal Peyer’s patches (PPs) contain a specialized conduit system that directs the flow of water absorbed across the intestinal epithelium. Notably, PP FRCs responded to conduit fluid flow via the mechanosensitive ion channel Piezo1. Disruption of fluid flow or genetic deficiency of Piezo1 on CCL19-expressing stroma led to profound structural alterations in perivascular FRCs and associated high endothelial venules. This in turn impaired lymphocyte entry into PPs and initiation of mucosal antibody responses. These results identify a critical role for conduit-mediated fluid flow in the maintenance of PP homeostasis and mucosal immunity.
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Data availability
The data that support the findings of this study are available from the corresponding author upon request. Microarrays are available on the Gene Expression Omnibus database with the accession number GSE135612.
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Acknowledgements
We thank the members of the Carroll laboratory and the Turley laboratory for comments and suggestions on the study. We thank B. Ludewig (Kantonsspital St. Gallen) for providing Ccl19Cre mice. This study was funded in part by Genentech and by the US National Institutes of Health (grant nos. F31 DK1055 to J.E.C.; R33 AI110164 and RO1 AI130307 to M.C.C.; RO1 DK074500 and R21 CA182598 to S.J.T.). We thank S. Jhunjhunwala and N. Lounsbury for management of sequencing files and data processing.
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J.E.C. designed and performed experiments, analyzed results and wrote the manuscript. M.B.B. performed experiments, analyzed results and contributed to the writing of the manuscript. E.G. performed experiments and analyzed results. M.C.C. and S.J.T. designed and supervised the study and contributed to the writing of the manuscript.
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S.J.T. and M.B.B are employed by Genentech.
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Supplementary Figure 1 Stromal cell numbers in PPs following treatments with PEG or amiloride.
(a,b) Flow cytometric analysis PP stromal cell populations (CD45−TER119−) following treatment with PEG or amiloride. Subpopulations are defined as FRC (PDPN+CD31−), LEC (PDPN+CD31+), BEC (PDPN−CD31+, and HEV (CD31+MAdCAM+). Mice were treated for a period of 3 days (a; n = 6 mice per group) or one week (b; n = 15 (Ctl, PEG) or 5 (Aml) mice per group). Representative FACS plots illustrate relative composition of FRCs (gp38+CD31−), LECs (gp38+CD31+), and BECs (gp38−CD31+). Data are graphed as relative cell counts. For all graphs, data represented as mean ± SEM. Groups were compared by one way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001. No statistical significance is indicated with ‘ns’.
Supplementary Figure 2 Blockade of fluid absorption equally affects B and T cell migration and total cellularity in the PP, but not myeloid cell populations.
(a) Frequency of total B and T lymphocytes resident in the PP, MLN, ILN, and spleen of control, PEG-treated, or amiloride-treated mice. (n = 9 (Ctl) or 12 (PEG, Aml) mice per group). (b) Flow cytometric analysis of myeloid cell populations from the PP of control or PEG-treated mice (n=8 (Ctl, PEG) or 4 (Aml) mice per group) (c) Flow cytometric analysis of mice following a 3-day treatment period with PEG or amiloride. Mice were additionally treated with either FTY720 or PBS on day 0 and day 2. Lymphocyte cellularity was analyzed from PP (n= 9 (Ctl, PEG) or 5 (Aml) mice per group) and ILN (n=5 mice per group). Data represented as mean ± SEM. Groups were compared by one way ANOVA. * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.005). No statistical significance is indicated with ‘ns’.
Supplementary Figure 3 Altered LN and PP cellularity is not a consequence of dehydration.
(a) Flow cytometric analysis of total lymphocyte counts from PP and LN of mice after the following treatments: control untreated, PEG for 3 days, PEG for 3 days then restored to normal drinking water for 12 hours (PEG > Water). (n=11 (Ctl, PEG) or 12 (PEG > Water) mice per group). (b) Flow cytometric analysis of total lymphocyte counts from PP and LN after the following treatments: control untreated, PEG for 3 days, amiloride for 3 days, PEG or amiloride for 3 days with twice daily subcutaneous injection of saline. (n = 13 (Ctl, PEG) or 8 (Aml) mice per group). Data represented as mean ± SEM. Groups were compared by one way ANOVA. * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.005). No statistical significance is indicated with ‘ns’.
Supplementary Figure 4 Conditional ablation of Piezo1 in FRCs.
(a) Transcriptional expression measured by RNAseq of Piezo1 in FRCs and HEV ECs isolated from the PP of control or PEG-treated mice. (n= 7 mice per group (FRC) or 4 mice per group (HEV EC)) (b) Validation of specific gene recombination in exons 20-23 of Piezo1. Genomic DNA isolated from PP stromal cells enriched by depletion of CD45+ cells using magnetic beads, primary FRCs expanded in culture for 5 days, or tail snips. Genotypes of mice indicated below each lane. Bands indicate the following: Intact gene flanked by flox sequences = 330 bp, knockout = 230bp, wildtype = 160bp. (c) Flow cytometric analysis of the distribution of eYFP-expressing cells isolated from the PP and inguinal LNs of Ccl19cre+ x ROSA26-eYFP mice. (n=3 mice per group). (d) Confocal microscopy of cultured PP FRCs. FRCs were enriched and culture expanded from Ccl19cre+ x ROSA26-eYFP mice. Representative of two independent experiments. (e) Transcriptional expression of Piezo1 in PP FRCs isolated from WT and Ccl19-P1cKO mice. FRCs were enriched and culture expanded before mRNA was measured by qPCR (n= 4 (Cre−) or 3 (Cre+) mice). Data represented as mean ± SEM. Groups were compared by unpaired two tailed Student’s t test. * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.005). No statistical significance is indicated with ‘ns’.
Supplementary Figure 5 Specific expression of Ccl19cre transgene in the FRC population.
(a,b) Confocal imaging analysis of the distribution of eYFP-expressing cells in the intestinal PP of Ccl19cre+ x ROSA26-eYFP mice. Representative of three experiments (a) FDCs are identified by positive staining with anti-CR1/CR2 (blue); MRCs are identified by positive staining with anti-RANKL (Red); FRCs are identified by the positive staining with anti-PDPN (grey) and absence of staining with anti-CR1/CR2 and anti-RANKL. Left image scale bar = 100um. Right images scale bar = 20um. (b) Representative confocal image of an HEV in cross-section. HEV endothelial cells are identified by positive staining with anti-MAdCAM (red). Scale bar = 10um.
Supplementary Figure 6 Altered LN and PP cellularity in Ccl19-P1cKO mice.
(a) Flow cytometric analysis of total stromal cell composition from the LN and PPs of Ccl19-P1cKO and Ccl19Cre − x Piezo1fl/fl (WT) mice. (n=3 mice per group). (b) Confocal imaging of lymphatic vessel architecture (identified by positive staining with anti-LYVE1) within the PP of control, PEG-treated or Ccl19-P1cKO mice. Representative of two independent experiments. (c) Flow cytometric analysis of B cell and T cell numbers from PP, MLN, and ILN of Ccl19-P1cKO and Ccl19Cre − x Piezo1fl/fl (WT) mice. Presented as relative cell counts (n=14 mice per group (PP, ILN) or 7 mice per group (MLN)). (d) Transcriptional expression measured by qPCR of Piezo1 in FRCs isolated from the LN and PP of untreated WT mice. (n=6 mice per group). For all graphs, data represented as mean ± SEM. Groups were compared by two way ANOVA (c) or unpaired two tailed Student’s t test (d). *P < 0.05, **P < 0.01, ****P < 0.0001. No statistical significance is indicated with ‘ns’.
Supplementary information
Supplementary Information
Supplementary Figs. 1–6.
Supplementary Video 1
Lymphocyte extravasation in PP of PEG-treated mouse. Mice were treated for 3 d with PEG in drinking water before imaging by multiphoton intravital microscopy. Labeled splenocytes (green) were adoptively transferred along with a Qtracker 655 vascular label (red) for visualization of the vasculature. Imaging began within 30–60 min of lymphocyte transfer. Time is shown in minutes and seconds. Video is representative of three independent experiments.
Supplementary Video 2
Lymphocyte extravasation in PP of amiloride-treated mouse. Mice were treated with amiloride 3 d before imaging by multiphoton intravital microscopy. Labeled splenocytes (green) were adoptively transferred along with a Qtracker 655 vascular label (red) for visualization of the vasculature. Imaging began within 30–60 min of lymphocyte transfer. Time is shown in minutes and seconds. Video is representative of two independent experiments.
Supplementary Video 3
Lymphocyte extravasation in PP of untreated mouse. Untreated mice were imaged by multiphoton intravital microscopy. Labeled splenocytes (green) were adoptively transferred along with a Qtracker 655 vascular label (red) for visualization of the vasculature. Imaging began within 30–60 min of lymphocyte transfer. Time is shown in minutes and seconds. Video is representative of three independent experiments.
Supplementary Video 4
Lymphocyte extravasation in PP of anti-MAdCAM1-treated mouse. Mice were treated with blocking anti-MAdCAM1 immediately before imaging by multiphoton intravital microscopy. Labeled splenocytes (green) were adoptively transferred along with a Qtracker 655 vascular label (red) for visualization of the vasculature. Imaging began within 30–60 min of lymphocyte transfer. Time is shown in minutes and seconds. Video is representative of two independent experiments.
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Chang, J.E., Buechler, M.B., Gressier, E. et al. Mechanosensing by Peyer’s patch stroma regulates lymphocyte migration and mucosal antibody responses. Nat Immunol 20, 1506–1516 (2019). https://doi.org/10.1038/s41590-019-0505-z
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DOI: https://doi.org/10.1038/s41590-019-0505-z
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