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. Author manuscript; available in PMC: 2020 Apr 14.
Published in final edited form as: Nat Immunol. 2019 Oct 14;20(11):1506–1516. doi: 10.1038/s41590-019-0505-z

Figure 7. FRCs respond to conduit fluid flow via Piezo1-mediated mechanosensation.

Figure 7.

(a) Representative images taken confocal microscopy of HEVs from Ccl19Cre − × Piezo1fl/fl (WT) and Ccl19-P1cKO mice. top and middle panels: HEV ECs and FRCs were marked by staining with anti-MAdCAM1 and anti-PDPN respectively. lower panel: Contact points between HEV perimeter and surrounding perivascular FRCs. Scale bars = 20um. Bar graph: Total quantification of FRC:HEV contact points from confocal imaging (n = 13 (WT) or 17 (cKO) mice). (b) Representative images taken by confocal microscopy of PP HEVs (cross-sectional orientation) from Ccl19Cre − × Piezo1fl/fl (WT) and Ccl19-P1cKO mice. Mice were in vivo labeled with anti-MAdCAM in alexafluor 488 by intravenous injection, then perfused with fixative and tissue sections from PPs were re-stained with anti-MAdCAM in PE. Cumulative data is quantified and displayed as percentage of pixels positive in MAdCAM-AF488 out of total MAdCAM+ pixels (% lumenal MAdCAM). Scale bars = 10um. (n = 18 (WT) or 16 (cKO) mice) (c) Flow cytometric analysis of adoptively transferred splenocytes accumulating in the PP or ILN one hour after transfer into Ccl19Cre− × Piezo1fl/fl (WT) and Ccl19-P1cKO recipients. (n = 6 (WT) or 7 (cKO) mice) (d) Flow cytometric analysis of total lymphocyte cellularity in the PP, ILN, and MLN of Ccl19Cre − × Piezo1fl/fl (WT) and Ccl19-P1cKO mice. Data are represented as fold change relative to untreated control mice. (n=14 mice per group). (e-g) Analysis of NP-specific humoral response following immunization against NP-CGG. Treatment groups include unimmunized WT mice (NI), immunized Ccl19Cre− × Piezo1fl/fl mice (WT), and immunized Ccl19-P1cKO mice (cKO). (n = 6 (NI), 9 (WT) or 11 (cKO) mice) (e) Representative flow cytometric analysis of NP-specific B cell percentages in the intestinal lamina propria. (Gated on CD45+, EpCAM−). (f) Quantification of total NP-specific B cell numbers in the intestinal lamina propria. (g) Anti-NP fecal IgA titers quantified by ELISA. For all graphs, data represented as mean ± SEM from at least three independent experiments. Groups were compared by one way ANOVA (f,g) or unpaired two tailed Student’s t test (a-d). *P < 0.05, **P < 0.01, ****P < 0.0001. No statistical significance is indicated with “ns”